WO2016150521A1 - Functionalised magnetic nanoparticle - Google Patents

Abstract

The present invention relates to functionalised magnetic nanoparticles and the process for preparing said functionalised magnetic nanoparticles. The invention also relates to the pharmaceutical composition comprising the functionalised magnetic nanoparticles. Further, the present invention relates to the therapeutic use of the functionalised magnetic nanoparticles and the pharmaceutical composition of the invention.

Description

FUNCTIONALISED MAGNETIC NANOP ARTICLE Field of the art

The present invention relates to the use of magnetic nanoparticles as carrier systems for therapeutic molecules. Particularly, the invention relates to multifunctional nanoparticles with a selective therapeutic effect for an efficient and controlled intracellular release of a drug.

State of the Art

The use of nano structures as carrier systems for therapeutic molecules has been explored during the last 20 years aiming to improve the therapeutic effect of the drugs and their administration, as well as, to reduce their side effects. Different types of nanostructures (metallic nanoparticles, polymeric core-shell nanoparticles or micelles) have been evaluated particularly in cancer therapy, whose vast side effects compromise the health of patients. Of particular interest are nanoparticles (NPs), which have a unique structure and characteristics such as a big surface to mass ratio and the ability to bind, absorb and carry other compounds such as drugs, nucleotides and proteins that widens their application fields and provides them with new or enhanced properties.

Nanoparticles for medical applications, and particularly for targeted cancer therapy, must be (1) non-toxic (2) with a good colloidal stability in physiological conditions; (3) easy to load with known amounts of therapeutic agents and targeting molecules and (4) able to release the cargo efficiently inside the cells. Therefore, the development of strategies for the functionalisation of nanoparticles is a crucial point for the future clinical use, and in particular, for improving anticancer therapies.

In spite of recent advances in nanotechnology-based therapies, the control of targeting to specific cells (i.e. cancer cells or cancer stem cells) still remains a challenge. This specificity would increase the efficiency of classical treatments and also avoid secondary effects.

In cancer therapy, one of the major obstacles is the toxicity and poor bioavailability of the chemotherapy drugs. In this sense, the use of nanoparticles can reduce the systemic toxicity of the anti-cancer drugs through targeted delivery. As an example, two of the drugs that have been traditionally employed in cancer therapy are doxorubicin (DOX), and gemcitabine (GEM). Despite positive results with these drugs in the clinic, classical chemotherapy still presents several problems. For example, doxorubicin has shown great efficacy in both solid and liquid tumours, but the emergence of drug resistance and several side effects such as heart muscle damage are important limitations for successful cancer treatment.

As a first approach at improving cancer therapy non-covalent functionalisation of nanoparticles, mainly through electrostatic or hydrophobic interactions has been explored intensively in the past due to the ease of application [R. Mejias et al., Biomaterials, 32 (2011) 2938-52; A. Z. Mirza & H. Shamshad, Eur. J. Med. Chem. 46 (2011) 1857-60]. However, this approach has some drawbacks. The most important limitation is the poor control on the release of the drug immobilised onto such nanoparticles. The drug release occurs as a passive process based on the high concentration of salts and bio molecules in vivo or on pH changes. This strategy is suitable for in vitro assays and using intra-tumoural injection in in vivo assays, but it should not be applied intravenously. Another drawback is the fact that neutral drugs, such as gemcitabine, cannot be immobilised electrostatically under physiological conditions.

X. Zeng et al. [Biomaterials 35 (2014) 1227-39] have recently disclosed that polyester- based hyperbranched dendritic- linear (HBDL)-based NPs carrying Dox can overcome microsomal glutathione transferase 1 (MGSTl)-mediated drug resistance in breast cancer cells. The authors also mention that the although DOX-HBDL NPs achieved enhanced drug accumulation in drug resistant cancer cells compared to drug-sensitive cells, the amount of drug delivered by the NPs was much lower compared to treatment with free drug.

Further, L.S. Jabr-Milane et al. [Cancer Treat. Rev. 34 (2008) 592-602] disclose multifunctional nanocarriers developed to enhance drug delivery and overcome MDR by either simultaneous or sequential delivery of resistance modulators (e.g., with P- glycoprotein substrates), agents that regulate intracellular pH, agents that lower the apoptotic threshold (e.g., with ceramide), or in combination with energy delivery (e.g., sound, heat, and light) to enhance the effectiveness of anticancer agents in refractory tumours. However, there is still a need in the art for alternative nanoparticle-based systems for the targeted delivery of drugs for medical applications, and particularly for cancer therapy.

Brief description of the invention The authors of the present invention have developed a functionalised magnetic nanoparticle with a selective therapeutic effect for the controlled and efficient release of a drug. Remarkably, the functionalised magnetic nanoparticle coupled with the linker design strategy according to the present invention, allows the intracellular release of the drug after an internal rearrangement without any chemical modification. The functionalised magnetic nanoparticles of the present invention have the required properties to be successfully employed in biomedical applications, and particularly in nanomedicine.

The invention also provides a process for preparing said multifunctional magnetic nanoparticle including an immobilisation strategy using tailored linkers. Therefore, in a first aspect, the invention is directed to a functionalised magnetic nanoparticle comprising a magnetic nanoparticle, a drug and a targeting agent, wherein the drug is covalently linked to the magnetic nanoparticle through a first disulfide linker, and wherein the targeting agent is covalently linked to the magnetic nanoparticle through a second disulfide linker, and wherein the first and the second linkers are the same or different.

In a second aspect, the invention is directed to a process for preparing the functionalised magnetic nanoparticle according to the invention, comprising the following steps:

a) activating the magnetic nanoparticle by introducing thiol moieties for the immobilisation of the drug and the targeting agent,

b) modifying the drug and the targeting agent by introducing a disulfide group and,

c) attaching the drug on the activated magnetic nanoparticle through a disulfide bond.

In a third aspect, the invention is directed to the functionalised magnetic obtainable by the process according to the invention. In a fourth aspect, the invention relates to the functionalised magnetic nanoparticles according to the invention for use as a medicament.

In a fifth aspect, the invention is directed to the functionalised magnetic nanoparticles according to the invention for use in the treatment of cancer. In a sixth aspect, the invention is directed to a pharmaceutical composition comprising the functionalised magnetic nanoparticles of the invention.

In another aspect, the invention relates to a pharmaceutical composition according to the invention for use as a medicament.

In another aspect, the invention relates to a pharmaceutical composition according to the invention for use in the treatment of cancer.

In another aspect, the invention is directed to the functionalised magnetic nanoparticle according to the invention for use in an in vivo method for diagnosing a disease characterised by presenting cells with differential expression of an antigen.

In another aspect, the invention is directed to a pharmaceutical composition according to the invention for use in an in vivo method for diagnosing a disease characterised by presenting cells with differential expression of an antigen.

In another aspect, the invention is directed to the use of a functionalised magnetic nanoparticle according to the invention as a contrast agent for imaging.

In another aspect, the invention is directed to the use of a pharmaceutical composition according to the invention as a contrast agent for imaging.

Figures

Figure 1 : General Scheme of the multifunctionalisation of DMSA-MNPs wherein a drug and a Nucant pseudopeptide are immobilised on the nanoparticle.

Figure 2: General Scheme of the multifunctionalisation of DMSA-MNPs wherein a drug and an anti-CD44 antibody are immobilised on the nanoparticle.

Figure 3: General scheme of the synthesis of the drug and Nucant derivatives.

Figure 4: Scheme of the drug release of DMSA-MNPs in presence of the reducing agent DTT. Figure 5: Characterisation of zeta potentials and hydrodynamic mono-functionalised and bi- functionalised DMSA-MNPs

Figure 6: Release kinetics of mono-functionalised DMSA-MNPs with DOX (A), GEM (B) and N6L (C) (1 μΜ DTT, filled squares and dashed line and 1 mM DTT, empty circles and solid line); (D) release kinetics of GEM from MNPs-GEM (empty squares, 1 mM GSH solid line and 1 μΜ GSH dashed line) and from MNPs-GEM-antiCD44 (filled triangles, 1 mM GSH solid line and 1 μΜ GSH dashed line).

Figure 7: (A) Prussian blue staining of Panc-1 cells (A, incubated with MNP-antiCD44 and B, incubated with MNP) and MDA-MB-231 cells (C, incubated with MNP-anti- CD44 antibody and D, incubated with MNP) (scale bar = 20 μπι). (B) ICP-MS results obtained for the incubation of Panc-1 and MDA-MB-231 with MNP-antiCD44 and MNP (n=3).

Figure 8: (A) Viability of Panc-1 cells control and treated with Free GEM (0.4, 1 and 4 μΜ), MNP-GEM-antiCD44 (4 μΜ GEM) and MNP-GEM (4 μΜ GEM); (B) HPLC profiles of GEM release.

Figure 9: UV-VIS spectra of DOX (A), GEM (B), and Nucant (C) immobilisation process.

Figure 10: UV-VIS spectra of Bradford's test of N6L immobilised on the nanoparticle (A), of N6L release (B) and of DOX release (C) .

Figure 11 : 1H NMR spectrum of 2-(pyridin-2-yldisulfanyl) ethanol (1);

Figure 12: 1H NMR spectrum of 4-nitrophenyl 2-(pyridin-2-yldisulfanyl) ethyl carbonate (2)

Figure 13: 1H NMR spectrum of doxorubicin derivative, DOX-S-S-Pyr (3)

Figure 14: ¹³C spectrum of doxorubicin derivative, DOX-S-S-Pyr (3)

Figure 15: 1H NMR spectrum of gemcitabine derivative, GEM-S-S-Pyr (4)

Figure 16: ¹³C spectrum of gemcitabine derivative, GEM-S-S-Pyr (4)

Detailed description of the invention

A. Functionalised magnetic nanoparticle The present invention is directed, in a first aspect, to a functionalised magnetic nanoparticle, hereinafter "the functionalised magnetic nanoparticle of the invention" comprising a magnetic nanoparticle, a drug and a targeting agent, wherein the drug is covalently linked to the magnetic nanoparticle through a first disulfide linker, and wherein the targeting agent is covalently linked to the magnetic nanoparticle through a second disulfide linker, and wherein the first and the second linkers are the same or different.

The term "magnetic nanoparticle" or "magnetic NP" or "MNP", as used herein, refers to a particle having a diameter ranging from about 1 to about 1000 nanometres and a saturation magnetisation (Ms) comprised between 20 and 200 emu/g, preferably between 50 and 90 emu/g. In a particular embodiment, the magnetic nanoparticle typically has an average particle diameter ranging from 2 to 50 nm, preferably from 4 to 25 nm, more preferably 15 nm. The average particle diameter is the average maximum particle dimension, it being understood that the particles are not necessarily spherical. The particle size may conveniently be measured using conventional techniques such as microscopy techniques, for example transmission electron microscopy.

In a particular embodiment, the magnetic nanoparticle has a spherical or substantially spherical shape. The shape may conveniently be assessed by conventional light or electron microscopy techniques.

In another particular embodiment, the magnetic nanoparticle is based on Fe, Co, Ni, metal oxides thereof or mixtures thereof. In a preferred embodiment, the magnetic nanoparticle is based on a metal oxide wherein the metal is Fe, Co, Ni or mixtures thereof. In a preferred embodiment, the magnetic nanoparticle is based on a metal oxide selected from gamma-Fe203 (maghemite), Fe₃04 (magnetite), CoO, C03O4 or NiO.

In another particular embodiment, the magnetic nanoparticle is based on stoichiometric ferrites, non-stoichiometric ferrites or doped ferrites.

In another preferred embodiment, the magnetic nanoparticle is based on ferrites having the general formula MFe₂0₄, wherein M represents a metal selected from: Co, Ni, Mg, Zn, Sr, or Mn. In a more preferred embodiment the magnetic nanoparticle is based on stoichiometric ferrites selected from MnFe₂04, CoFe₂04, ZnFe₂04, NiFe₂04, MgFe₂04, SrFei₂0i9 or BaFe^Oig. In another preferred embodiment the magnetic nanoparticle is based on non- stoichiometric ferrites selected from Fe3__xM_x0₄ wherein M is a transition elements selected from Cr, Mn, Co, Ni and Zn; Mn Fe₂C"4, MnaZri(i__a)Fe₂04 and Ni_aZn(i__a)Fe₂04 being a<l . In a more preferred embodiment, the magnetic nanoparticle is based on

In a particular embodiment, the first and/or the second disulfide linker comprise an organosulfur moiety covalently bound to a disulfide amide moiety.

In the context of the present invention, the term "organosulfur moiety" refers to a moiety containing at least a thiol group. In a particular embodiment, the organosulfur compound is a dimercaptosuccinic moiety.

In the context of the present invention the disulfide amido moiety is any moiety which comprises at least an amide group (-NHCOO-) and at least a disulfide group (S-S).

In a particular embodiment the disulfide amido moiety is selected from:

In the context of the present invention, the term "drug" refers to a chemical substance used in the treatment, cure, or prevention of a disease or condition, e.g., cancer, etc. The chemical nature of the drug can vary broadly, e.g. it can be a small molecule, a peptide, and so on. Although different and numerous kinds of drugs can be used within the context of the invention, in a particular embodiment, the present invention contemplates that the drug is selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor, an anthracycline, and a nucleoside analogue.

As used herein, the term "alkylating agent" or "alkylating antineoplasic agent" refers to an agent that mediates the transfer of an alkyl group from one molecule to DNA. The alkyl group may be transferred as an alkyl carbocation, a free radical, a carbanion or a carbene (or their equivalents). Alkylating agents are used in chemotherapy to damage the DNA of cancer cells. The alkylating agents are generally separated into six classes:

- nitrogen mustards, such as mechlorethamine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, etc.;

- ethylenamine and methylenamine derivatives, including altretamine, thiotepa and the like;

- alkyl sulfonates, such as busulfan, etc.;

- nitrosoureas, such as carmustine, lomustine, etc.;

triazenes, such as dacarbazine, procarbazine, temozolomide, etc.; and

- platinum- containing antineoplastic agents, such as cisplatin, carbop latin and oxaliplatin, which are usually classified as alkylating agents, although they do not alkylate DNA, but cause covalent DNA adducts by a different means, etc. As used herein, the term "antimetabolite" refers to a chemical that inhibits the use of a metabolite, which is another chemical that is part of normal metabolism. Such substances are often similar in structure to the metabolite that they interfere with, such as the antifolates that interfere with the use of folic acid. The presence of antimetabolites can have toxic effects on cells, such as halting cell growth and cell division, so these compounds are used as chemotherapy for cancer. Anti-metabolites masquerade as a purine or a pyrimidine, preventing their incorporation into DNA during the S phase (of the cell cycle), stopping normal development and division. They also affect RNA synthesis. However, because thymidine is used in DNA but not in RNA (where uracil is used instead), inhibition of thymidine synthesis via thymidylate synthase selectively inhibits DNA synthesis over RNA synthesis. Antimetabolites may be selected from:

- purine analogues, such as azathioprine, mercaptopurine, thioguanine fludarabine pentostatin, cladribine, etc.; - pyrimidine analogues, such as gemcitabine, 5-fluorouracil (5FU), floxuridine (FUDR), cytosine arabinoside (cytarabine), 6-azauracil (6-AU), etc.; or antifolates, such as methotrexate, pemetrexed, proguanil, pyrimethamine, trimethoprim, etc.

Preferably, the antimetabolite is gemcitabine.

As used herein, the term "topoisomerase inhibitor" refers to an agent designed to interfere with the action of topoisomerase enzymes (topoisomerase I and II). It is thought that topoisomerase inhibitors block the ligation step of the cell cycle, generating single and double stranded breaks that harm the integrity of the genome. Introduction of these breaks subsequently leads to apoptosis and cell death. Illustrative, non- limitative examples of topoisomerase inhibitors include etoposide, teniposide, topotecan, irinotecan, diflomotecan or elomotecan.

As used herein, the term "anthracycline" refers to a class of drugs (CCNS or cell-cycle non-specific) used in cancer chemotherapy derived from strains of Streptomyces bacteria. Anthracyclines have four mechanisms of action:

1. Inhibition of DNA and RNA synthesis by intercalating between base pairs of the DNA/RNA strand, thus preventing the replication of rapidly-growing cancer cells.

2. Inhibition of topoisomerase II enzyme, preventing the relaxing of supercoiled DNA and thus blocking DNA transcription and replication.

3. Creation of iron-mediated free oxygen radicals that damage the DNA, proteins and cell membranes.

4. Induction of histone eviction from chromatin that deregulates DNA damage response, epigenome and transcriptome. In a particular embodiment the drug is an anthracycline selected from doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin and mitoxantrone. In a preferred embodiment, the drug is an anthracycline, preferably, doxorubicin.

In a particular embodiment the drug may be charged or neutral, fluorescent or not.

The functionalised MNP of the invention further comprises a targeting agent, which is covalently linked to the MNP through a disulfide bond. In the context of the present invention, the term "targeting agent" refers to an entity that specifically recognises or binds to sites or regions on a target cell. The term "target cell", as used herein, refers to a diseased or cancerous cell. Targeting agents suitable for the present invention comprise, or consist of, without limitation, antibodies, peptides, aptamers and pseudopeptides. In preferred particular embodiment, the protein is an antibody (Ab). The term "antibody" is used herein in the sense of its capacity to bind specifically to an antigen and thus, it refers to a molecule having such capacity. Included within said term are: an intact antibody that binds specifically to the target antigen; and an antibody fragment that binds specifically to the target antigen. As used herein, the term "intact antibody" refers to an immunoglobulin molecule capable of specific binding to its cognate target, including a target, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one binding recognition site (e.g., antigen binding site), including a site located in the variable region of the immunoglobulin molecule. An antibody includes an antibody of any class, namely IgA, IgD, IgE, IgG (or sub-classes thereof), and IgM, and the antibody need not be of any particular class. In a preferred embodiment, the antibody is an IgG.

As used herein, the term "antibody fragment" refers to functional fragments of antibodies, such as Fab, Fab', F(ab')₂, Fv, single chain (scFv), heavy chain or fragment thereof, light chain or fragment thereof, a domain antibody (DAb) (i.e., the variable domain of an antibody heavy chain (VH domain) or the variable domain of the antibody light chain (VL domain)) or dimers thereof, VH or dimers thereof, VL or dimers thereof, nanobodies (camelid VH), and functional variants thereof, fusion proteins comprising an antibody, or any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site of a desired specificity. An antibody fragment may refer to an antigen binding fragment. In a preferred embodiment, the antibody fragment is a VH or domain antibody or DAb. In another preferred embodiment, the antibody fragment is a scFv. In another preferred embodiment, the antibody fragment is a nanobody.

Techniques for the preparation and use of the various antibodies are well known in the art. For example, fully human monoclonal antibodies lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice or from phage display libraries.

For use in the instant invention, the antibody is preferably an antibody which specifically binds to an antigen exposed on the cell surface. Illustrative, non- limitative examples of antigens suitable in the context of this invention include tumour antigens, such as HER2, EGFR, PSA, PSMA, CEA, CD (cluster of differentiation) markers such as CD20 (marker of B-cells), CD4 (T-helper cells), CD8 (T-suppressor cells), CD34 (hematopoietic stem cells), CD44, etc., and bacterial antigens, such as flagellin (H antigen), cell wall lipopolysaccharide (O antigens), and capsular polysaccharide (K antigen) in different bacteria strains including Escherichia coli and Smith surface antigen {Staphylococcus aureus). In a preferred embodiment, the antibody is an anti- CD44 antibody.

In another particular embodiment, the targeting agent comprises, or consists of, a peptide. As used herein, the term "peptide" refers to a short chain of amino acid monomers linked by peptide bonds. The peptide will comprise at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, or at least 70 amino acids. Suitable for the purposes of this invention are peptides with, among others, capacity to penetrate a cell, to provoke signalling, or to bind to a target.

In a preferred embodiment, the peptide is selected from the group consisting of a cell- penetrating peptide, a signalling peptide and a target binding peptide.

In another preferred embodiment, the peptide is a cell-penetrating peptide. In another preferred embodiment, the peptide is a signalling peptide. In another preferred embodiment, the peptide is a target binding peptide. As used herein, the term "cell- penetrating peptide" or "CPP" refers to a short peptide that facilitate cellular uptake of various molecular cargo, particularly, of ferritin nanoparticles. CPPs typically have an amino acid composition that either contains a high relative abundance of positively charged amino acids such as lysine or arginine or has sequences that contain an alternating pattern of polar/charged amino acids and non-polar, hydrophobic amino acids. These two types of structures are referred to as polycationic or amphipathic, respectively. A third class of CPPs are the hydrophobic peptides, containing only apolar residues, with low net charge or have hydrophobic amino acid groups that are crucial for cellular uptake. Numerous CCPs are known in the art, examples of which can be found at Tables 1 and 2 in Veldhoen et al. (2008, Int J Mol Sci 9: 1276-320), which are incorporated herein by reference.

As used herein, the term "signalling peptide" refers to a peptide with capacity of provoking cell signalling, such as agonist peptides of cells receptors. Examples of signalling peptides include, without limitation, CNN intercellular signalling peptide, signaling lymphocytic activation peptide, and neuropeptides, such as a-melanocyte- stimulating hormone (a-MSH), galanin-like peptide, cocaine-and-amphetamine- regulated transcript (CART), neuropeptide Y, agouti-related peptide (AGRP), β- endorphin, cholecystokinin, dynorphin, enkephalin, galanin, ghrelin, growth-hormone releasing hormone, neurotensin, neuromedin U, and somatostatin.

As used herein, the term "target binding peptide" refers to a peptide comprising a target binding region. Amino acid sequences suitable for binding target molecules include consensus sequences of molecular recognition well known in the art. These include without limitation:

sequences containing the RGD motif to target integrins, preferably the RGDLXXL (SEQ ID NO: 1) sequence, wherein "X" is any amino acid, such as TTYTASARGDLAHLTTTHARHLP (SEQ ID NO: 2),

RGDLATLRQLAQEDGVVGVR (SEQ ID NO: 3), SPRGDLAVLGHKY (SEQ ID NO: 4), CRGDLASLC (SEQ ID NO: 5), etc.;

- the LINK domain from TSG-6 is the preferred sequence to target hyaluronan, but also domains from hyaluronan receptors RHAMM and CD44 can be used; - the laminin receptor binding peptide [YIGSR (SEQ ID NO : 6)];

- VEGF receptor binding peptide (VRBP) (SEQ ID NO: 7);

- pro-gastrin-releasing peptide (ProGRP) to target gastrin-releasing peptide receptor;

PHSRN motif from fibronectin to target alpha(5)beta(l) integrin fibronectin receptor (SEQ ID NO: 8);

- NGR that binds aminopeptidase N (CD 13). In a particular embodiment the targeting agent is an aptamer. Preferably, the targeting agent is an aptamer selected from a peptide aptamer and a DNA aptamer.

As used herein, the term "peptide aptamer" refers to a short variable peptide domain that is attached at both ends to a protein scaffold, and that binds to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool, but natural aptamers also exist in riboswitches. Aptamers can be used for both basic research and clinical purposes as macromolecular drugs. As such, peptide aptamers are proteins that are designed to interfere with other protein interactions inside cells. The variable loop length is typically composed of ten to twenty amino acids, and the scaffold may be any protein which has good solubility and compacity properties. Currently, the bacterial protein Thioredoxin-A is the most used scaffold protein, the variable loop being inserted within the reducing active site, which is a Cys-Gly-Pro-Cys loop (SEQ ID NO: 9) in the wild protein, the two Cys lateral chains being able to form a disulfide bridge. Peptide aptamer selection can be made using different systems, including the yeast two-hybrid system, phage display, mRNA display, ribosome display, bacterial display and yeast display.

The term "DNA aptamer", as used herein, refers to a short strand of DNA that has been engineered through repeated rounds of selection to bind to specific molecular targets, such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. DNA aptamers are useful in biotechno logical and therapeutic applications as they offer molecular recognition properties that rival that of the commonly used biomolecule, antibodies, and elicit little or no immunogenicity in therapeutic applications. The selection of DNA aptamers is well-known in the art using techniques such as systematic evolution of ligands by exponential enrichment (SELEX). In another particular embodiment, the targeting agent comprises, or consists of, a pseudopeptide. As used herein, the term "pseudopeptide" refers to analogues of peptide or proteins that mimic the biological activities of natural peptides or proteins. In the context of the present invention pseudopeptides may be peptide analogues obtained by replacing one or more amino acids of the L series with one or more of the corresponding D series, or peptides exhibiting a modification at the level of at least one of the peptide bonds, such as the retro, inverso, retro-inversi, carba and aza bonds. The pseudopeptide will comprise at least 2 amino acids, at least 3 amino acids, at least 4 amino acids, at least 5 amino acids, at least 10 amino acids, at least 15 amino acids, at least 20 amino acids, at least 30 amino acids, at least 40 amino acids, at least 50 amino acids, at least 60 amino acids, or at least 70 amino acids. Suitable for the purposes of this invention are pseudopeptides with, among others, capacity to penetrate a cell, to provoke signalling, to bind to a target.

Examples of pseudopeptides include HB-19, Carfilzomib (PR-171), Oprozomib (PR- 047), Delanzomib (CEP- 18770), Bortezomib, Epoxomicin and Nucant.

In a preferred embodiment, the pseudopeptide is a Nucant pseudopeptide. The term "Nucant pseudopeptide" refers to a sequence of the pseudo-tripeptide

Pro-Arg (moieties are all in L configuration) coupled to the Lys residues of a polypeptide template containing Aib (2-aminoisobutyric acid): Ac-Lys-Aib-Gly-Lys- Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-Lys-Aib-Gly-CONH2 (SEQ ID NO: 10). N6L is a member of the pseumultivalent Nucant pseudopeptides family, that present pentavalently, such as N3, or hexavalently, such as N6, N6L, N7. The pseudopeptide Nucant (N6L) is a peptide that targets tumours and exhibit antitumoural activity in various human tumour cell lines derived from mammary, colorectal carcinoma, melanoma, glioblastomas, and lymphoma, displaying entiangiogenic activity in various in vitro and in vivo experiments. N6L binds nucleolin, which is a protein overexpressed in the membrane of cancer cells, and nucleophosmin, and can enter the cell nucleus to induce apoptosis. In a preferred embodiment, the pseudopeptide is the N6L Nucant pseudopeptide.

In a preferred embodiment the functionalised magnetic nanoparticle comprises a magnetic nanoparticle, a drug and a targeting agent, wherein the magnetic nanoparticle is an iron oxide nanoparticle; wherein the drug is gemcitabine; wherein the targeting agent is an anti-CD44 antibody; wherein gemcitabine is covalently linked to the iron oxide nanoparticle and wherein the anti-CD44 antibody is covalently linked to the iron oxide nanoparticle through a disulfide amide moiety.

In another preferred embodiment, the functionalised magnetic nanoparticle of the invention comprises a magnetic nanoparticle, a drug and a targeting agent, wherein the magnetic nanoparticle is iron oxide nanoparticle; wherein the drug is gemcitabine or doxorubicin; and wherein the targeting agent is N6L Nucant pseudopeptide, wherein the gemcitabine or the doxorubicin is covalently linked to the iron oxide nanoparticle and wherein the targeting agent is covalently linked to the iron oxide nanoparticle through a disulfide amide moiety.

In another particular embodiment, the functionalised magnetic nanoparticle further comprises an imaging agent. As used herein, the term "imaging agent" refers to a chemical compound that is designed to allow the localisation of the target cell, wherein the cell is preferably a diseased or cancerous cell. Non- limitative examples of imaging agents suitable for the purposes of this invention include radionuclides, fluorophores and magnetic contrast agents.

In a preferred embodiment, the imaging agent comprises, or consists of, a radionuclide. To this end, appropriate radionuclides are loaded as agents for diagnostic imaging methods, such as radioimmunodiagnostics, positron emission tomography (PET). Non- limitative examples of radionuclides include gamma-emitting isotopes, for example, ^99mTc, ¹²³I, and ^mIn, which can be used in radio scintigraphy using gamma cameras or single-photon emission computed tomography, as well as positron emitters, for example, ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, ⁸⁶Y, ¹²⁴I, ²¹³Bi and ²¹¹ At, that can be used in PET or beta emitters, such as ¹³¹I, ⁹⁰Y, ^99mTc, ¹⁷⁷Lu, and ⁶⁷Cu". In another preferred embodiment, the imaging agent comprises, or consists of, a fluorophore. The term "fluorophore", as used herein, refers to a fluorescent chemical compound that can re-emit light upon light excitation. Fluorescent dyes include, without limitation, Cy3, Cy2, Cy5 and FITC.

In another preferred embodiment, the imaging agent comprises, or consists of, a magnetic contrast agent. The term "magnetic contrast agent" or "MRI agent", as used herein, refers to a group of contrast media used to improve the visibility of internal body structures in magnetic resonance imaging (MRI). Examples of MRI agents include, without limitation, gadolinium-based compounds, superparamagnetic iron oxide (SPIO) and ultra-small superparamagnetic iron oxide (USPIO), iron platinum-based compounds and manganese based compounds.

B. Process for preparing the functionalised magnetic nanoparticle Further, the present invention provides a process for preparing the functionalised magnetic nanoparticle, hereinafter "the process for preparing the functionalised magnetic nanoparticle of the invention", comprising the following steps:

a) activating the magnetic nanoparticle by introducing thiol moieties for the immobilisation of the drug and the targeting agent,

b) modifying the drug and the targeting agent by introducing a disulfide group and,

c) attaching covalently the drug and the targeting agent to the activated magnetic nanoparticle of step (a) by a disulfide bond.

In the context of the present invention, the terms "magnetic nanoparticle", "drug" and "targeting agent" mentioned in the above process are understood as previously defined in section A related to the functionalised magnetic nanoparticle of the invention, and their particular and preferred embodiments apply equally here. According to step a) of the defined process, the magnetic nanoparticle is activated to permit the subsequent drug and targeting agent immobilisation. The activation step is performed in order to assure a fixed and controlled amount of free thiol functions on the magnetic nanoparticle. This step is represented in the general schemes of figures 1 and 2, which are not intended to limit the scope of the invention. According to step a) of the above process, the magnetic nanoparticle is activated by the introduction of thiol groups on the surface for the immobilisation of the drug and the targeting agent. In the context of the present invention, to introduce the thiol goups on the magnetic nanoparticle, the magnetic nanoparticle is previously coated with an organosulfur compound containing at least a carboxylic group. In the context of the present invention, the term "organosulfur compound" refers to a compound having at least a thiol group (-SH) and at least a carboxylic group (-COOH). In a particular embodiment, the organosulfur compound is dimercaptosuccinic acid (DMSA).

In the context of the present invention, the magnetic nanoparticle coated with the organosulfur compound is bound to an aminothiol compound. In the context of the present invention, the term "aminothiol compounds" refers to compounds having at least an amine group and at least a thiol group. The term "thiol group" refers to a carbon-bound sulfhydryl group (-C-SH).

In a particular embodiment the aminothiol compounds are selected from cysteamine, 3- amino-propane-1 -thiol, 4-amino-butane-l -thiol, and l-amino-2-mercapto-ethane-l,2- diol. Preferably, the aminothiol compound coating the magnetic nanoparticle is cysteamine.

The amine group of the aminothiol compound react with a carboxylic group of the organosulfur compound coating the magnetic nanoparticle giving as result an amide moiety. The free thiol groups of the resulting amide moiety are ready to react.

In a particular embodiment, the MNP coated with the organosulfur compound is incubated with neutralized cysteamine hydrochloride in the presence of EDC and NHS at room temperature. The DMSA-MNP may be incubated with neutralized cysteamine hydrochloride in the presence of EDC and NHS at room temperature, preferably the cysteamine hydrochloride may be previously neutralized with NaOH (see step 1 in figure 1).

Therefore, in a particular embodiment in step a) of the described process, the magnetic nanoparticle is activated through the introduction of an organosulfur moiety covalently bound an amide-thiol moiety.

In a particular embodiment, the organosulfur moiety is a dimercaptosuccinic moiety. In another particular embodiment the amide-thiol moiety is selected from:

According to step b) of the above process, the drug and the targeting agent are modified by introducing a disulfide group in their chemical formula to promote the reaction with the sulfhydryl groups of pre-activated coated magnetic nanoparticle. In a particular embodiment, the drug and the targeting agent are modified with a disulfide reactive. In a preferred embodiment the drug and the targeting agent are modified with a pyridyldisulfide group. More preferably, the disulfide reactive is 2,2'-dipyridyldisulfide (see figure 3).

In a particular embodiment, the drug to be modified is selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline. In a preferred embodiment, the drug to be modified is an anthracycline selected from doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin and mitoxantrone. More preferably, the drug to be modified is doxorubicin.

In another particular embodiment the drug is a nucleoside analogue selected from gemcitabine, didanosine, vidarabine, BCX4430, cytarabine, emtricitabine, lamivudine, zalcitabine, abacavir, aciclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine and trifluridine. More preferably, the drug is gemcitabine.

In a particular embodiment, the drug is modified through the following steps (figure 3):

1. Synthesis of 2-(pyridin-2-yldisulfanyl) ethanol (1) by a simple disulfide exchange reaction of aldrithiol with 2-mercaptoethanol.

2. Synthesis of 4-nitrophenyl 2-(pyridin-2-yldisulfanyl)ethyl carbonate (2) by the reaction of bis(4-nitrophenyl) carbonate with the primary alcohol 2-(pyridin-2- yldisulfanyl) ethanol obtained in step 1.

3. Synthesis of the drug derivative through the carbonate moiety by the nucleophilic substitution of the 4-nitrophenol leaving group.

Preferably, the drug to be modified according to the above mentioned steps is gemcitabine or doxorubicin. In this particular embodiment, a modification of the drug to introduce a pyridyldisulfide group is required to promote the reaction with the activated magnetic nanoparticle obtained in step a) of the above defined process for preparing the functionalised magnetic nanoparticle.

In another particular embodiment, the targeting agent to be modified is selected from proteins, peptides, pseudopeptides, aptamers, proteins and antibodies.

In another particular embodiment the targeting agent to be modified is an antibody. The introduction of free thiol groups onto the antibody may be achieved by the reaction between Traut's reagent or 2-iminothiolane and the amine groups of the antibody. In an antibody molecule, it is possible to distinguish at least two types of amino groups exposed to the medium: (i) the terminal amino groups and (ii) the ε-amino moiety of lysine residues. While terminal amino groups have a pK around 7-8, ε-amino groups of Lys residues have a pK close to 10. At pH values less than 8.0, the Ab amino terminal groups are the most reactive. As the amino terminal moieties are located in the Fab region where antigen recognition takes place, the Ab modification at this pH condition could contribute to a lower activity of the Ab after its functionalisation While at pH values higher than 8.0, ε-amino groups of Lys residues are more reactive and as the majority of the lysine residues are located in the Fc portion, the modification should occur preferentially in the Fc portion. Reactions at pH > 8 are particularly preferred in the context of the present invention. In the step c) of the above defined process, the drug and the targeting agent are covalently attached to the free thiol groups of the activated magnetic nanoparticle of step (a) by a disulfide bond. The functionalisation of the nanoparticle is achieved by the formation of disulfide bonds between the activated nanoparticle and the modified drug, and between the activated nanoparticle and the modified targeting agent. In the context of the present invention, the presence of disulfide bonds between MNPs and drugs will permit the controlled release of the drug. In a particular embodiment the release of the drug is produced under intracellular reducing conditions. In particular, the disulfide bond can be broken by specific reducing agent such as endogenous glutathione (GSH) present in millimolar concentrations (0.5-1.0 mM) in the cells but in micromolar concentrations in blood plasma and the extracellular medium. Without wishing to be bound to any theory, it is believed that the free thiol generated upon disulfide cleavage is able to attack the carbonate moiety favoured by the formation of a ring, leaving the drug unaltered (see scheme in figure 4). In a particular embodiment, the free thiol generated upon disulfide cleavage attacks the carbonate moiety favoured by the formation of a leaving five member ring group. As result, the drug is released and its activity is not affected. This is particularly useful when the functionalised magnetic nanoparticle is internalised by the cell, where the drug is released intracellularly.

Moreover, it has been shown by clinical studies that tumour tissue is often higher in glutathione content than normal tissue. By designing the functionalisation with a disulfide bond, the attached molecule will be released only under highly reducing environment such as the tumour cells' intracellular environment.

C. Pharmaceutical Compositions

The present invention provides a pharmaceutical composition, hereinafter "the pharmaceutical composition of the invention", comprising the functionalised magnetic nanoparticle of the invention.

The functionalised magnetic nanoparticle has been described in detail in the context of the functionalised magnetic nanoparticle of the invention, and its particular and preferred embodiments apply equally to the pharmaceutical composition of the invention.

Examples of pharmaceutical compositions include any solid composition (tablets, pills, capsules, pellets, etc.) or liquid composition (solutions, suspensions or emulsions) for oral, topical or parenteral administration (sterile solutions, suspensions or lyophilized products in a suitable unit dosage form). They can contain active ingredients or other materials with biomedical applications; conventional excipients known in the art, such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, corn starch, calcium phosphate, sorbitol or glycine; lubricants for preparing tablets, for example magnesium stearate; disintegrating agents, for example starch, polyvinylpyrrolidone, sodium glycolate of starch or micro crystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate. The formulations mentioned will be prepared using common methods such as those described or referred to in the Spanish and United States Pharmacopeias and in similar reference texts.

The pharmaceutical composition provided by the present invention may be administered to a subject by any suitable route of administration, such as, for example, via intratumoural or parenteral.

The term "parenteral" as used herein includes intravenous, intraperitoneal, intramuscular, or subcutaneous administration. The intravenous form of parenteral administration is generally preferred. In addition, the pharmaceutical composition provided by the present invention may suitably be administered by pulse infusion, e.g. with declining doses of the therapeutic ferritin nanoparticle. Preferably the dosing is given by injections, most preferably intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. In another embodiment, the pharmaceutical composition provided by the present invention may be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate unit dosage form. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyetheylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and/or gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound (e.g., a therapeutic ferritin nanoparticle) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

In a particular embodiment, said pharmaceutical composition is administered via intravenous or intratumoural. Adequate excipients can be used, such as bulking agents, buffering agents or surfactants. The mentioned formulations will be prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.

It is especially advantageous to formulate the pharmaceutical compositions, namely, oral or parenteral compositions, in dosage unit form for ease administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound (the functionalised magnetic nanoparticle of the invention) calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals. Active compounds (agents) will typically be administered once or more times a day for example 1 , 2, 3 or 4 times daily, with typical total daily doses in the range of from 0.001 to 1,000 mg/kg body weight/day, preferably about 0.01 to about 100 mg/kg body weight/day, most preferably from about 0.05 to 10 mg/kg body weight/day. The pharmaceutical composition will be formulated in order to contain the desired amount, such as a therapeutically effective amount of the agent present in the ferritin nanoparticle.

The pharmaceutical compositions provided by the present invention can be included in a container, pack, or dispenser together with instructions for administration. The pharmaceutical compositions provided by the present invention may be used with other drugs to provide a combination therapy. The other drugs may form part of the same composition, or be provided as a separate composition for administration at the same time or at different time.

The pharmaceutical compositions provided by the present invention will be useful in the treatment of medical conditions, such as diseases capable of benefiting from the treatment with a therapeutic agent, specially, for treating tumour diseases or cancers. In a particular embodiment the pharmaceutical compositions provided by the present invention are suitable in the treatment of cancer selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, bladder cancer, endometrial cancer, kidney cancer, breast cancer, stomach cancer, non-Hodgkin's lymphoma, thyroid cancer, soft- tissue sarcoma, metastatic breast cancer, ovarian cancer, prostate cancer and rectal cancer.

D. Therapeutic uses

The smaller dimensions of the nanoparticles of the invention mean that they have unique physical properties. Their small size turns them into systems ideal for use in biological applications.

The capacity of the nanoparticles of the invention to generate an energy transfer allows application in the treatment of tumours by means of hyperthermia. Magnetic hyperthermia is one of the few methods having the potential theoretical possibility of causing localised damage in the tumour without damaging the adjacent healthy tissue. The magnetic nanoparticles convert the electromagnetic energy into heat when they are exposed to external radio frequency (RF) fields such that the ferromagnetic and superparamagnetic nanoparticles of the invention can be applied for obtaining a controlled heat in carcinogenic tumours, opening up new possibilities in cancer therapy. Also, upon being internalised, the drug is released under the reducing intracellular conditions. The functionalised magnetic nanoparticle provided by the present invention may be used for the treatment of diseases.

Thus, in another aspect, the present invention relates to a functionalised magnetic nanoparticle according to the invention for use as a medicament. In another aspect, the present invention relates to a functionalised magnetic nanoparticle according to the invention for use in the treatment of cancer.

This aspect may be alternatively formulated as a use of a functionalised magnetic nanoparticle according to the invention in the treatment of cancer, or as a functionalised magnetic nanoparticle according to the invention for use in the manufacture of a medicament for the treatment of cancer. Further, the invention also contemplates an in vivo method for treating cancer comprising administering the functionalised magnetic nanoparticle according to the invention.

The functionalised magnetic nanoparticle has been described in detail in the context of the functionalised magnetic nanoparticle of the invention, and its particular and preferred embodiments apply equally to the therapeutic uses of the invention.

According to the invention, the functionalised magnetic nanoparticle is delivered to the target cell. Further, upon being internalised, the drug is released under the reducing intracellular conditions.

As used in the context of the therapeutic uses of the invention, the term "target cell" refers to the particular cell to which the targeting agent of the functionalised magnetic nanoparticle binds.

In a particular embodiment, the target cell is a mammalian cell. In a preferred embodiment, the mammalian cell is a human cell. Non- limitative examples of human cells include, without limitation, somatic cells, germ cells and stem cells. Advantageously, in a particular embodiment, the target cells, i.e., the cells which the agent is delivered to, are malignant cells, including tumour cells. Thus, in a particular embodiment, the target cell is a malignant cell. In a preferred embodiment, the target cell is a tumour cell. As used herein, the term "tumour cell" or "cancer cell" refers to cells that grow and divide at an unregulated, quickened pace. The term "cancer" or "tumour" or "tumour disease", as used herein, refers to a broad group of diseases involving unregulated cell growth and which are also referred to as malignant neoplasms. Cancers usually share some of the following characteristics: sustaining proliferative signalling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and eventually metastasis. Cancers invade nearby parts of the body and may also spread to more distant parts of the body through the lymphatic system or bloodstream. Cancers are classified by the type of cell that the tumour cells resemble, which is therefore presumed to be the origin of the tumour. These types include:

Carcinoma: Cancers derived from epithelial cells. This group includes many of the most common cancers, particularly in the aged, and include nearly all those developing in the breast, prostate, lung, pancreas, and colon.

Sarcoma: Cancers arising from connective tissue (i.e. bone, cartilage, fat, nerve), each of which develop from cells originating in mesenchymal cells outside the bone marrow.

Lymphoma and leukaemia: These two classes of cancer arise from hematopoietic (blood-forming) cells that leave the marrow and tend to mature in the lymph nodes and blood, respectively. Leukaemia is the most common type of cancer in children accounting for about 30%.

Germ cell tumour: Cancers derived from pluripotent cells, most often presenting in the testicle or the ovary (seminoma and dysgerminoma, respectively).

Blastoma: Cancers derived from immature "precursor" cells or embryonic tissue. Blastomas are more common in children than in older adults. In a particular embodiment the functionalised magnetic nanoparticles provided by the present invention may be used as a medicament in the treatment of pancreatic cancer, lung cancer, colon cancer, bladder cancer, endometrial cancer, kidney cancer, breast cancer, stomach cancer, non-Hodgkin's lymphoma, thyroid cancer, soft-tissue sarcoma, metastatic breast cancer, ovarian cancer, prostate cancer and rectal cancer.

According to the present invention, the functionalised magnetic nanoparticle is suitable for the treatment of a disease, wherein said agent is indicated for treating said disease. As used herein, the term "treatment" or "therapy" can be used indistinctly and refer to clinical intervention in an attempt to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disease, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.

The term "subject" or "individual" refers to a member of a mammalian species, and includes but is not limited to domestic animals, and primates including humans; the subject is preferably a male or female human being of any age or race.

Non- limitative examples of treatments suitable in the context of the present invention include radiotherapy, which uses specific radiotherapeutic agents, and cytotoxic chemotherapy, which uses specific chemotherapeutic agents. Examples of chemotherapy and/or radiotherapy agents include radionuclides and drugs, respectively. Radionuclides and drugs are conventional and well-known by the person skilled in the art, and examples have been described previously and are incorporated here by reference.

In the particular embodiment of radiotherapy, the functionalised magnetic nanoparticle further comprises an imaging agent which is a radionuclide. In particular, the radionuclides useful for radiotherapy, alpha-emitting, beta-emitting and gamma- emitting radionuclides are particularly useful. Radionuclides suitable for use in radiotherapy are well-known by the skilled person. Illustrative examples that are useful in the context of the present invention include, without limitation, alpha emitters, such as ²¹³Bi and ²¹¹ At; beta emitters, such as ⁹⁰Y, ^99mTc, ¹⁷⁷Lu, and ⁶⁷Cu; and gamma- emitters, such as ¹³¹I.

In the context of the drugs useful for chemotherapy, drugs suitable for use in chemotherapy are well-known by the skilled person. Illustrative examples that are useful in the context of the present invention include, without limitation, an alkylating drug, such as nitrogen mustards, cyclophosfamide, alkyl sulfonates, temozolomide, and cisplatin; an antimetabolite, such as azathioprine, 5-fluorouracil, and methotrexate; a topoisomerase inhibitor, such as irinotecan and etoposide; and an anthracycline, such as doxorubicin and mitoxantrone.

Therefore, in a particular embodiment, the chemotherapy and/or radiotherapy agent is selected from the group consisting of a radionuclide and a drug. In a preferred embodiment, the chemotherapy and/or radiotherapy agent is a radionuclide. In another preferred embodiment, the chemotherapy and/or radiotherapy agent is a drug. In a more preferred embodiment, the agent is an antitumoural drug.

It is common knowledge in the art which drugs are indicated for the treatment of a particular type of cancer, and while radionuclides are indicated for the treatment of virtually all cancers, the indication of drugs, also known as anti-cancer or anti-tumoural drugs, is somewhat more restricted. By way of illustrative example, a relation of different types of cancers and drugs that are indicated for the treatment of said types of cancers is given in Table 1.

Table 1

Relation of cancers and drugs that are indicated for their treatment

These therapeutic applications will comprise the administration of a therapeutically effective amount of the functionalised magnetic nanoparticle of the invention. The term "therapeutically effective amount", as used herein, refers to the amount of said functionalised magnetic nanoparticle according to the invention which is required to achieve an appreciable cure or killing of cells of said disease. For the administration to a subject in need thereof of a functionalised magnetic nanoparticle according to the invention, said functionalised magnetic nanoparticle will be formulated in a suitable pharmaceutical composition. The particulars of said pharmaceutical composition have been discussed in the context of the pharmaceutical compositions of the invention discussed.

Thus, in another aspect, the present invention relates to a pharmaceutical composition according to the invention for use as a medicament.

In another aspect, the present invention relates to a pharmaceutical composition according to the invention for use in the treatment of cancer. This aspect may be alternatively formulated as a use of a pharmaceutical composition according to the invention in the treatment of cancer, or as a pharmaceutical composition according to the invention for use in the manufacture of a medicament for the treatment of cancer. Further, the invention also contemplates an in vivo method for treating cancer comprising administering the pharmaceutical composition according to the invention.

The pharmaceutical composition according to the invention has been described in detail previously, and its particular and preferred embodiments apply equally to the therapeutic uses of the invention.

The present invention also contemplates treatment by hyperthermia as explained previously.

E. Diagnostic uses

It will be immediately understood by the person skilled in the art that the functionalised magnetic nanoparticle of the invention may be used for imaging by means of magnetic resonance techniques. Also, in the particular embodiment where the functionalised magnetic nanoparticle of the invention further comprises an imaging agent, said functionalised magnetic nanoparticle can be used in in vivo delivering said imaging agent to a target cell, or for visualizing a target cell. Thus, the functionalised magnetic nanoparticles of the invention find application in diagnostics, especially in in vivo diagnosis by imaging techniques. Therefore, in another aspect, the invention contemplates the use of the functionalised magnetic nanoparticle according to the invention for use in an in vivo method for diagnosing a disease characterised by presenting cells with differential expression of an antigen. The functionalised magnetic nanoparticle has been described in detail in the context of the functionalised magnetic nanoparticle of the invention, and its particular and preferred embodiments apply equally to the therapeutic uses of the invention.

The term "disease characterised by presenting cells with differential expression of an antigen", as used herein, refers to a disease wherein the cells comprised in the diseased area, tissue or organ express an antigen which is not substantially expressed by healthy cells.

In a particular embodiment, the disease characterised by presenting cells with differential expression of an antigen is selected from cancer, a cardiovascular disease and a bacterial infection. In a preferred embodiment, the disease characterised by presenting cells with differential expression of an antigen is cancer. In another preferred embodiment, the disease characterised by presenting cells with differential expression of an antigen is a cardiovascular disease. In another preferred embodiment, the disease characterised by presenting cells with differential expression of an antigen is a bacterial infection. The term "cancer" has been described in detail in the context of the therapeutic uses of the invention, and its particular and preferred embodiments apply equally to the diagnostic uses of the invention.

The term "cardiovascular disease", as used herein, refers to a class of diseases that involve the heart or blood vessels. Examples of cardiovascular diseases include ischemic heart disease (IHD), stroke, hypertensive heart disease, rheumatic heart disease (RHD), aortic aneurysms, cardiomyopathy, atrial fibrillation, congenital heart disease, endocarditis, and peripheral artery disease (PAD).

The term "bacterial infection", as used herein, refers to illness caused by infection with pathogenic bacteria. Examples of bacterial diseases include, without limitation, acinetobacter infections, bacterial pneumonia, mycobacterium tuberculosis, bacterial vaginosis, urinary tract infection, endocarditis, pneumonia, bacterial gastroenteritis, salmonellosis, peritonitis, septicemia, or bacterial meningitis.

The functionalised magnetic nanoparticle of the invention can act as contrast agents in systems for obtaining images by means of magnetic resonance (Magnetic Resonance Imaging), or the analysis of biological samples by means of optical and electron spectroscopy, improving on the viewing efficiency. The particular techniques that are suitable for imaging magnetic nanoparticles are well known by the person skilled in the art.

In another particular embodiment, the functionalised magnetic nanoparticle of the invention further comprises an imaging agent.

The term "imaging agent" has been described in detail in the context of the functionalised magnetic nanoparticle of the invention, and its particular and preferred embodiments apply equally to the diagnostic uses of the invention.

As it is common knowledge in the art, the person skilled in the art will immediately know which imaging techniques are suitable for visualising each imaging agent.

The present invention also contemplates the pharmaceutical compositions of the invention for use in in vivo methods of diagnosis, as described previously.

Further, the functionalised magnetic nanoparticle of the invention comprising at least a targeting agent and a drug can be used as a tracer of a target cell. The magnetism of the nanoparticle in the functionalised magnetic nanoparticle of the invention allows the detection of tumour cells, cancer cells or infected cells.

The nanoparticles of the invention also have application in the transport and/or immobilisation, as well as in the controlled release of active ingredients in a biological medium. The nanoparticles of the invention can be used as the tracers of drug release instead of radioactive materials used, which allow monitoring the release of a drug through the measurement of magnetic property variations, eliminating the harmful effects of radiation. Additionally, they can be used in vaccination guns as an alternative to the vaccine injectors which are commonly compressed air or gas (particularly helium), causing pain and leaving marks on the skin. The injection power would in this case be provided by applying a magnetic field, which would cause the nanoparticles to speed up in their passage through the epidermis. Thus, in another aspect, the present invention relates to the use of the functionalised magnetic nanoparticle according to the invention as a contrast agent for imaging.

In another aspect, the present invention relates to the use of the pharmaceutical composition according to the invention as a contrast agent for imaging.

Examples

Specific embodiments of the invention which in no case must be considered limiting are presented below.

Example 1 : Pre-activation of DMSA-MNPS

DMSA-MNPs are first modified with cysteamine hydrochloride to introduce thiol moieties (thiolated DMSA-MNPs). 5 mL of MNP at 2.4 mg Fe/mL are incubated overnight at room temperature with 50 μιηοΐ of cysteamine hydrochloride/g Fe, previously neutralized by 1 equivalent of NaOH, 150 μιηοΐ of EDC/g Fe and 75 μιηοΐ of NHS/g Fe. After 16 h, the sample is washed by cycles of centrifugation and redispersion in milliQ water 3 times. The presence of sulfhydryl groups introduced on MNP is quantitatively measured by reaction with 2,4-dinitrothiocyanatebenzene (DNTB)

The preactivated DMSA-MNPs are stable at physiological pH. DMSAMNPs thiolated present a zeta potential of -54.5 ± 2.1 mV and a hydrodynamic diameter of 59.0 ± 1.8 nm whereas DMSA-MNPs present a zeta potential of -60.1 ± 2.1 mV and a hydrodynamic diameter of 53.1 ± 0.9 nm.

Example 2: Synthesis of drugs derivatives

2.1 Synthesis of 2-(Pyridin-2-yldisulfanyl)ethanol (I)

To a solution of aldrithiol (300 mg, 1.36 mmol) in MeOH (1.5 mL) under Ar, 2- mercaptoethanol (53 μί, 0.75 mmol) is added slowly and stirred for 16 h. Then, the solvent is evaporated in vacuum and the residue purified by flash chromatography (CH₂Cl₂/AcOEt 5: 1) to obtain compound 1 (Figure 3) as a colorless oil in 86% yield; 1H NMR (300 MHz, CDC13) δ 8.51 (d, J = 4.3 Hz, 1H), 7.58 (td, J = 8.0, 1.7 Hz, 1H), 7.40 (d, J = 8.0 Hz, 1H), 7.17 - 7.13 (m, 1H), 5.72 (bs, 1H), 3.80 (dd, J = 10.4, 6.5 Hz, 2H), 2.97 - 2.94 (m, 2H), (figure 11). 2.2 Synthesis of 4-Nitrophenyl 2-(pyridin-2-yldisulfanyl)ethyl carbonate (2)

To a solution of compound 1 (100 mg, 0.53 mmol), and bis(4-nitrophenyl) carbonate (241 mg, 0.79 mmol) in CH₂C1₂ (2 mL) under Ar, DIPEA (158 pL, 0.79 mmol) is added and stirred for 5 h. The mixture is washed with water, and the organic phase dried with MgS04. After solvent evaporation, the residue is purified by flash chromatography (Hexane/AcOEt 4: 1 and then 2: 1) to obtain compound 2 (Figure 3) as a colorless oil 67% yield; 1H NMR (300 MHz, CDC1₃) δ 8.50 (d, J = 4.8 Hz, 1H), 8.28 (d, J = 9.1 Hz, 2H), 7.72 - 7.59 (m, 2H), 7.38 (d, J = 9.1 Hz, 2H), 7.15 - 7.10 (m, 1H), 4.57 (t, J = 6.4 Hz, 2H), 3.16 (t, J= 6.4 Hz, 2H), (figure 12). 2.3.1 Synthesis of Doxorubicin derivative, DOX-S-S-Pyr (3)

To a solution of compound 2 (10 mg, 0.028 mmol) and doxorubicin hydrochloride (12 mg, 0.020 mmol) in DMF (1 mL) under N₂, DIPEA (8 μΕ, 0.028 mmol) is added at room temperature and stirred for 16 h. Then, the solvent is evaporated and the residue is purified by flash chromatography (eluent: CH₂Cl₂/MeOH 20: 1) to obtain compound 3 (Figure 3) as red solid in 90% yield; 1H NMR (500 MHz, CDC1₃) δ 13.94 (s, 1H), 13.18 (s, 1H), 8.40 (d, J= 3.8 Hz, 1H), 8.00 (d, J= 7.6 Hz, 1H), 7.76 (t, J= 8.0 Hz, 1H), 7.70 (d, J = 7.5 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.37 (d, J = 8.5 Hz, 1H), 7.07 (t, J = 1H), 5.50 b(s, 1H), 5.26 (dd, J= 3.7, 2.0 Hz, 1H), 5.15 (d, J= 8.8 Hz, 1H), 4.74 b(s, J= 21.4 Hz, 2H), 4.56 (bs, 1H), 4.42 - 4.34 (m, 1H), 4.21 - 4.08 (m, 2H), 4.06 (s, 3H), 3.81 (m, 1H), 3.62 (bs, 1H), 3.22 (dd, J = 18.8, 1.5 Hz, 1H), 3.14 - 2.86 (m, 5H), 2.33 (d, J = 14.6 Hz, 1H), 2.15 (dd, J = 15.0, 3.6 Hz, 1H), 1.81 (m, 2H), 1.48 - 1.42 (m, 2H), 1.29 (d, J = 6.5 Hz, 3H); ¹³C NMR (126 MHz, CDC1₃) δ 213.9, 187.0, 186.6, 161.0, 160.1, 156.2, 155.6, 155.2, 149.8, 149.4, 137.3, 135.7, 135.4, 133.6, 133.6, 120.9, 120.8, 119.9, 119.8, 118.4, 111.5, 111.4, 100.9, 69.7, 69.0, 67.4, 65.6, 63.5, 56.7, 53.4, 47.0, 37.5, 35.6, 33.9, 30.0, 29.7, 28.3, 17.0; MS (ESI): m/z (%)757(100), (figures 13 and 14).

2.3.2 Synthesis of Gemcitabine derivative, GEM-S-S-Pyr (4)

To a solution of compound 2 (25 mg, 0.071 mmol) and gemcitabine chlorhydrate (24 mg, 0.09 mmol) in DMF (1.5 mL) under Ar, DIPEA (18 μί, 0.09 mmol) and DMAP (catalytic amount) are added and stirred for 16 h. Then, the solvent is evaporated in vacuum and the residue purified by flash chromatography (CH₂Cl₂/MeOH 15: 1) to obtain compound 4 (Figure 3) in 38% yield as a color less oil; 1H NMR (500 MHz, CDCls) δ 8.44 - 8.43 (m, 1H), 7.70 - 7.64 (m, 2H), 7.52 (d, J = 6.8 Hz, 1H), 7.11 (ddd, J = 6.7, 4.9, 1.5 Hz, 1H), 6.23 (bs, 2H), 5.80 (d, J = 7.5 Hz, 1H), 5.33 (bs, 1H), 4.50 - 4.38 (m, 3H), 4.13 (d, J = 7.5 Hz, 1H), 4.04 (d, J = 12.0 Hz, 1H), 3.84 (d, J = 12.0 Hz, 1H), 3.13 - 3.02 (m, 2H); ¹³C NMR (126 MHz, CDC1₃) δ 165.8, 159.2, 155.6, 153.5, 149.8, 149.6, 137.4, 121.1, 120.0, 95.7, 78.7, 78.7, 72.7, 66.8, 65.4, 59.6, 36.5; MS (ESI): m/z (%)239 (23), 477 (M⁺+H, 100), 499 (M⁺+Na, 2); HRMS (ESI) calculated for C17H19F2N4O6S2 (M⁺+H) 477.0708, found 477.0708; HRMS (ESI) calculated for Ci₇Hi₈N406F₂NaS2 (M⁺+Na) 499.0547, found 499.0528, (figures 15 and 16).

Example 3: Covalent attachment of drug derivatives to thiolated DMSA-MNPs

3.1 Reaction between thiolated DMSA-MNPs and Doxorubicin derivative (3)

1 mL of aqueous suspension of thiolated DMSA-MNPs at 2.4 mg Fe/mL is mixed with 240 μΕ of DOX-S-S-Pyr (3) solution at 500 μΜ in DMF (0.012 μιηοΐ) during 16 h at 37°C. After this time, 20

of brine are added and the sample is centrifuged 10 min at 5000xg. From the collected supernatants, the covalently immobilised DOX onto thiolated DMSA-MNPs is determined by quantification of the 2-pyridinethione released ( πιαχ = 343 nm, 8343nm = 8080 L.mor'.crn ¹, figure 9A). Finally the sample is redispersed in lmL of MilliQ water. 3.2 Reaction between thiolated DMSA-MNPs and Gemcitabine derivative (4)

1 mL of aqueous suspension of thiolated DMSA-MNPs at 2.4 mg Fe /mL is mixed with 240 μΐ, of GEM-S-S-Pyr (4) solution at 500 μΜ in DMF (0,012 μιηοΐ) during 16 h at 37°C. After reaction, 20 μΐ_^ of brine are added and the sample centrifuged 10 min at 5000xg. From the collected supernatants, the covalently immobilised GEM onto thiolated DMSA-MNPs is determined by quantification of the 2-pyridinethione released ( πιαχ = 343 nm, 8343nm = 8080 L.mor'.crn ¹, figure 9B). Finally, the sample is redispersed in lmL of MilliQ water.

The reaction of modified DOX and GEM with pre-activated DMSA-MNPs led to stable colloidal formulations of functionalised DMSA-MNPs with 32 μιηοΐ DOX/g Fe and 30 μιηοΐ GEM/g Fe, respectively. In this case, a slight difference is observed in the zeta potential and the hydrodynamic diameter of these particles compared with DMSA- MNPs. The immobilisation yields are 64% for DOX and 60% for GEM with loads of 32 μιηοΐ DOX/g Fe and 30 μιηοΐ GEM/g Fe, respectively. Zeta potentials and hydrodynamic diameters obtained for each formulation are showed in figure 5.

Example 4: Synthesis of targeting agent derivative Example 4.1 Synthesis of MNP-N6L conjugate.

1 mL of aqueous suspension of thiolated DMSA-MNPs at 2.4 mg Fe/mL is mixed with 84 mL of Nucant-S-S-Pyr (5) at 200 μΜ in water (0,0168 μιηοΐ) during 16 h at RT. The reaction mixture is centrifuged and washed with brine to eliminate electrostatically immobilised Nucant and then with water. From the collected supernatant the covalently immobilised Nucant onto DMSA-MNPs is determined by quantification of the 2- pyridinethione released ( rnax =343 nm, 8343 nm = 8080 L mol^"1 cm^"1, figure 9C).

The yield of immobilisation is 70% (5 mmol N6L/g Fe). The zeta potential of the sample is -41.9 ± 3.3 mV at pH 7.4 with a hydrodynamic diameter of 102.2 ± 0.4 nm.

Example 4.2 Synthesis of MNP-antiCD44 conjugate

The introduction of free thiol groups onto the antibody is achieved by the reaction between Traut's reagent or 2-iminothiolane and the amine groups of the antibody [R.R. Traut et al, Biochemistry. 12 (1973) 3266-3273]. The Ab modification is carried out at pH values higher than 8.0 employing a 0.01 M HEPES, 0.15 M NaCl, pH 8.2 solution. After the reaction, the immobilised antibody is quantified by Bradford assay. The standard load obtained of covalently linked anti-CD44 antibody is 30 mg /g Fe (87%>), corresponding to around 1 antibody molecule per nanoparticle. The remaining pyridyldisulfide groups are blocked with 3-mercaptopropionic acid. No release of immobilised Ab is observed at this step, due to the higher reactivity of pyridyldisulfide groups. After that, the MNP-antiCD44 is purified by gel filtration thought a sepharose CL-6B column using 0.01 M sodium phosphate, pH 7.4 solution. The sodium phosphate MNP-GEM-antiCD44 suspension is stable for weeks stored at 4°C without noticeable precipitation (zeta potential of -43.0 ± 1.1 mV and hydrodynamic diameter of 82.6 ± 1.5 nm, figure 5). Example 5: Bi-functionalisation of thiolated DMSA-MNPs

Example 5.1: Covalent immobilisation of GEM or DOX and N6L on thiolated DMSA- MNPs (MNP-DOX-N6L and MNP-GEM-N6L)

1 mL of aqueous suspension of MNP-DOX at 2.4 mg Fe/mL is mixed with 84 μΕ of N6L-S-S-Pyr (5) solution at 200 μΜ in water (0.0168 μιηοΐ) during 16 h at RT. After reaction, 20 μΐ_^ of brine are added and the sample is centrifuged 10 min at 5000xg three times to eliminate any electrostatically bound N6L. UV-Vis absorption of supernatants is checked to quantify the N6L covalently bound. The same protocol is employed for the synthesis of MNP-GEM-N6L. Zeta potentials and hydrodynamic diameters obtained for each formulation are showed in figure 5.

Example 5.2: Covalent immobilisation of GEM and anti-CD44 antibody on thiolated DMSA-MNPs

A gemcitabine derivative (0.36 μιηοΐ, 30 μιηοΐ/g Fe) is added to react with sulfhydryl pre-activated MNP (5 mL at 2.4 mg Fe /mL) 0.36 μιηοΐ, 30 μιηοΐ/g Fe. The covalently immobilised GEM is determined by quantification of the 2-pyridinethione released during the reaction

= 343 nm, 8343nm = 8080 M.cm^"1) (figure 9B).

Then, remaining sulfhydryl groups of MNP-GEM are activated as follows: 5 mL of aqueous suspension of sulfhydryl activated MNP-GEM at 2.4 mg Fe /mL is mixed with 60 of 2-aldrithiol solution at 5 mM in DMSO (0.3 μιηοΐ, 25 μιηοΐ/g Fe) during 2 h at 40°C. After reaction, 200 μΐ_^ of brine are added and the sample centrifuged 10 min at lOOOOxg and redispersed in 5mL of 0.01 M sodium phosphate, pH 7.4.

Finally, anti-CD44 antibody is immobilised on MNP-GEM following the same protocol described above for immobilisation on MNP. Example 6: In vitro drug release studies

Example 6.1: In vitro drug release of MNP-GEM and MNP-DOX

The cumulative drugs release experiments are carried out using two different conditions in order to evaluate the stimuli-response behaviour of functionalised DMSA-MNPs toward reducing environment. The release of drugs, from the functionalised DMSA- MNPs is carried out under physiological conditions (pH 7.4 and 37°C) using two different concentrations (ΙμΜ and ImM) of reducing agent 1 ,4-Dithiothreitol (DTT) to mimic the extracellular and intracellular conditions. For each experiment, 2.4 mg of functionalised MNPs (or 4,8 mg in the case of the MNP-GEM and MNP-DOX) are dissolved in 1 mL of 10 mM phosphate buffer at pH 7.4 containing ΙμΜ of DTT, or 10 mM phosphate buffer pH 7.4 containing ImM DTT and incubated at 37°C. The amount of each drug released is determined by different methods at regular time intervals (figures 6 A, B and C). The amount of DOX released is analysed by measuring the absorbance of the sample at 495 nm with UV-Vis spectrophotometer (figure IOC). The amount of released GEM is analysed by HPLC using a C-18 column, mobile phase water/acetonitrile 80/20, at flow rate of 0.3 mL/min, measuring the absorbance at 270 nm (figure 8B). The amount of released N6L is analysed using Bradford's method by measuring the absorbance of the sample at 595 nm with the UV- Visible spectrophotometer (figure 10B).

Example 6.2: In vitro drug release of MNP-GEM and MNP-GEM-antiCD44

The cumulative drug releases, from the MNP-GEM and MNP-GEM-antiCD44 are carried out under physiological conditions (pH 7.4 and 37°C) using two different concentrations of glutathione (GSH) as reducing agent (1 μΜ and 1 mM of GSH to mimic the extracellular and intracellular conditions, respectively). For each experiment, 4.8 mg of MNP-GEM and MNP-GEM-antiCD44 are dissolved in 1 mL of 0.01 M phosphate buffer at pH 7.4 containing either 1 μΜ of GSH or 1 mM GSH and incubated at 37°C. The amount of GEM released is analysed at regular time intervals by HPLC using a C-18 column, mobile phase water/acetonitrile 80/20, at flow rate of 0.3 mL/min, measuring the absorbance at 270 nm. The percentage of GEM released is calculated from a standard calibration curve of free drug solution (figure 6D).

Both formulations show similar standard release (96-98% release when treated with 1 mM GSH (as intracellular environment) after 6-8 h while only 3-5% of the cargo is released with 1 μΜ GSH (as extracellular environment) after 6-8 h (figure 6). These results show that the release of GEM from MNP-GEM and MNP-GEM-antiCD44 is selective and strongly dependent on the reducing environment so that it takes place mostly inside the cells and is not affected by the presence of the antibody. Example 6.3: In vitro drug release of GEM-MNP-antiCD44

Cells culture Panc-1 and MDA-MB231 cell lines (American Type Culture Collections, Manassas, VA, USA) are grown as monolayer in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal bovine serum (FBS) at a final concentration of 10%, 2 mM L- glutamine, 0.25 /zg/mL fungizone and 100 units of penicillin and 100 μg/mL of streptomycin. Cell lines are maintained at 37°C in a humidified atmosphere consisting of 75% air and 5% C0₂ in an incubator.

Sterilisation of MNP

Magnetic iron oxide nanoparticles (MNP) sterilisation is carried out before cell incubation. 500 μΐ of MNP stock is dispersed by sonication for 5 minutes and then MNP are mixed with medium containing 10% FBS until desired concentration. The resulting sample is filtered through a 0.22 μιη Millex-GP filter (Merck-Millipore Darmstadt, Germany) and sonicated again for 1 minute.

Targeting to cancer cells with MNP-antiCD44

To determine the specific targeting of MNP-antiCD44 for Panc-1 and MDA-MB231 cell lines, cells are seeded at 2.5 10⁴ cells per well in 500 μΐ of DMEM containing 10%> FBS. After 24 h, the growth medium is removed and cells are then incubated 4 h at 4 °C in the presence of MNP and MNP-antiCD44 (0.2 mg Fe/mL, Ab 30 μg/mg Fe, 4 μΜ Gemcitabine). After incubation, cells are washed three times with PBS. Prussian blue staining of iron, processing for electron microscopy and inductively coupled plasma mass spectrometry (ICP-MS) are performed to investigate the specific binding of MNP - antiCD44 to cells expressing CD44 receptor (figure 7).

Prussian blue staining

For Prussian blue staining, cells are seeded on 12 mm square glass coverslips (Maienfeld GmbH & Co. KG, Germany) placed into the wells. Briefly, the cells are washed twice with PBS (AMRESCO, Ohio, USA) and fixed with 4% paraformaldehyde solution for 30 min at room temperature. Again, cells are washed twice with PBS, and are then incubated with a 1 : 1 mixture of 4% potassium ferrocyanide and 4% hydrochloric acid (Prussian blue staining solution) for 15 minutes at room temperature and washed with distilled water three times. The counterstaining is done for cytoplasm with neutral red 0.5% (Panreac Quimica S.L.U) for 2 minutes at room temperature and then washed with distilled water several times. After drying the cells, a cover slip is mounted by using DePeX (SERVA Electrophoresis GmbH) and finally, the cells are observed using light microscopy (Leica DMI3000B, Leica Microsystems, Germany). All experiments are carried out in triplicate (figure 7).

Inductively coupled plasma mass spectrometry (ICP-MS)

For ICP-MS, the cells are washed twice with PBS (AMRESCO, Ohio, USA), trypsinized with 200 of 0.25% w/v trypsin solution and are then incubated 5 minutes at 37 °C. When a single cell suspension is obtained, 2 ml of complete media is added. The resultant solution is transferred to a sterile 15 mL conical centrifuge tube and is spun down at 1200 rpm for 10 minutes. The supernatant is discarded carefully, then cells are resuspended in 5 mL of fresh complete media and 100 is collected to count cell number. The cell suspension is centrifuged again at 1200 rpm for 10 minutes and the supernatant is discarded carefully. 300 of HC1 at 37% is added to the cell pellet, and the resultant suspension is sonicated for 30 minutes at 40°C. Finally 2700 μΐ of bi- distilled water is added to the sonicated suspension and the iron concentration is determined by measuring the sample in ICP-MS NexION 300XX (Perkin Elmer).

Example 7: In vitro cytotoxicity assays

To assess cell death, Panc-1 cells are cultured on a 24-well plate at a density of 2.5 x 10⁴ cells per well in 500 μΐ of complete medium. After 24 h, the growth medium is removed and cells are then incubated 4 h at 4°C in the presence of different concentrations of free Gemcitabine (4, 1 and 0.4 μΜ), MNP-GEM and MNP-GEM- antiCD44 (0.2 mg Fe/mL, 4 μΜ Gemcitabine). After incubation, cells are washed three times with PBS and then maintained in DMEM supplemented with 10% FBS at 37°C and 5% C0₂ incubator. After 72 h, the medium is replaced with DMEM supplemented with 10% FBS, and 10% of Resazurin dye (1 mg/ml PBS). Cells are maintained at 37°C and 5% C0₂ incubator for 3 hours. A Synergy H4 microplate reader is used to determine the amount of Resazurin reduced by measuring the absorbance of the reaction mixture (excitation 570 nm, emission 600 nm). 600 μΐ of 10% of Resazurin dye is added to empty wells as a negative control. The obtained results are shown in figure 8. The viability of the cells is expressed as the percentage of absorption of treated cells in comparison with control cells (without nanoparticles). All experiments are carried out in triplicate. All the data obtained are plotted and statistically analysed using the software package GraphPad Prism version 5.0 for Windows. All samples are compared using a one-way ANOVA and Bonferroni post-hoc test (*P < 0.05, **P < 0.01, and ***P < 0.001). Only significant differences among the samples are indicated in the charts.

The obtained results brings both the confirmation of the selectivity of antibody functionalised MNP for Panc-1 cell line and the confirmation of the drug release mechanism only within the cell and not out of the cell during the initial incubation time. Also significant differences between the MNP-GEM-antiCD44 and free drug doses (0.4 and 1 μΜ of GEM) are observed 3 days after the drug treatment (#P < 0.05). No significant differences between the MNP-GEM-antiCD44 (4 μΜ of GEM) and the higher free drug dose (4 μΜ of GEM) are observed after 3 days of treatment.

Claims

A functionalised magnetic nanoparticle comprising a magnetic nanoparticle, a drug and a targeting agent, wherein the drug is covalently linked to the magnetic nanoparticle through a first disulfide linker, and wherein the targeting agent is covalently linked to the magnetic nanoparticle through a second disulfide linker, and wherein the first and the second linker are the same or different.

The functionalised magnetic nanoparticle according to claim 1 wherein the first and/or the second disulfide linker comprises an organosulfur moiety covalently bound to a disulfide amido moiety.

The functionalised magnetic nanoparticle according to claim 2 wherein the organosulfur moiety is a dimercaptosuccinic moiety.

The functionalised magnetic nanoparticle according to claim 2 wherein the disulfide amido moiety is selected from:

and

5. The functionalised magnetic nanoparticle according to any of claims 1, 2, 3 and 4 wherein the targeting agent is selected from a peptide, a pseudo peptide and an antibody.

The functionalised magnetic nanoparticle according to claim 5 wherein the antibody is an anti-CD44 antibody.

The functionalised magnetic nanoparticle according to claim 5 wherein the pseudo peptide is a Nucant pseudo peptide.

The functionalised magnetic nanoparticle according to any of claims 1 to 7 wherein the drug is selected from an anthracycline and a nucleoside analogue

The functionalised magnetic nanoparticle according to claim 8 wherein the anthracycline is selected from doxorubicin, daunorubicin, epirubicin, idarubicin, valrubicin and mitoxantrone.

10. The functionalised magnetic nanoparticle according to claim 8 wherein the nucleoside analogue is selected from gemcitabine, didanosine, vidarabine, BCX4430, cytarabine, emtricitabine, lamivudine, zalcitabine, abacavir, aciclovir, entecavir, stavudine, telbivudine, zidovudine, idoxuridine and trifluridine.

11. The functionalised magnetic nanoparticle according to any of claims 1 to 10 wherein the magnetic nanoparticle is selected from Fe, Co, Ni, their corresponding oxides and mixtures thereof.

12. The functionalised magnetic nanoparticle according to any of claims 1 to 11 comprising a magnetic nanoparticle, a drug and a targeting agent, wherein the magnetic nanoparticle is an iron oxide nanoparticle; wherein the drug is gemcitabine; wherein the targeting agent is an anti-CD44 antibody; wherein gemcitabine is covalently linked to the iron oxide nanoparticle and wherein the anti-CD44 antibody is covalently linked to the iron oxide nanoparticle through a disulfide amide moiety.

13. The functionalised magnetic nanoparticle according to any of claims 1 to 11 comprising a magnetic nanoparticle, a drug and a targeting agent, wherein the magnetic nanoparticle is iron oxide nanoparticle; wherein the drug is gemcitabine or doxorubicin; and wherein the targeting agent is N6L Nucant pseudopeptide, wherein the gemcitabine or the doxorubicin is covalently linked to the iron oxide nanoparticle and wherein the targeting agent is covalently linked to the iron oxide nanoparticle through a disulfide amide moiety.

14. The functionalised magnetic nanoparticle according to any of claims 1 to 13 further comprising an imaging agent.

15. The functionalised magnetic nanoparticle according to claim 14, wherein the imaging agent is selected from a radionuclide, a fluorophore and a magnetic contrast agent.

. A process for preparing the functionalised magnetic nanoparticle as defined in any of claims 1 to 15, comprising:

a) activating the magnetic nanoparticle by introducing thiol moieties for the immobilisation of the drug and the targeting agent,

b) modifying the drug and the targeting agent by introducing a disulfide group and,

c) attaching the drug on the activated magnetic nanoparticle through a disulfide bond.

17. The process according to claim 16 wherein the magnetic nanoparticle is activated through the introduction of organosulfur moiety covalently bound an amide-thiol moiety.

18. The process according to claim 17 wherein the organosulfur moiety is a dimercaptosuccinic moiety.

19. The process according to claim 17 wherein the amide-thiol moiety is selected from:

20. A functionalised magnetic nanoparticle obtainable by the process as defined in claims 16-19.

21. Functionalised magnetic nanoparticle according to any of claims 1 to 15 and 20 for use as a medicament.

22. Functionalised magnetic nanoparticle according to any of claims 1 to 15 and 20 for use in the treatment of cancer.

23. Functionalised magnetic nanoparticle for use according to claim 22 wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, bladder cancer, endometrial cancer, kidney cancer, breast cancer, stomach cancer, non-Hodgkin's lymphoma, thyroid cancer, soft-tissue sarcoma, metastatic breast cancer, ovarian cancer, prostate cancer and rectal cancer.

24. Pharmaceutical composition comprising the nanoparticle according to any of claims 1 to 15 and 20.

25. Pharmaceutical composition according to claim 24 for use as a medicament.

26. Pharmaceutical composition according to claim 24 for use in the treatment of cancer.

27. Pharmaceutical composition for use according to claim 26 wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, bladder cancer, endometrial cancer, kidney cancer, breast cancer, stomach cancer, non-Hodgkin's lymphoma, thyroid cancer, soft-tissue sarcoma, metastatic breast cancer, ovarian cancer, prostate cancer and rectal cancer.

28. Functionalised magnetic nanoparticle according to any of claims 1 to 15 and 20, or pharmaceutical composition according to claim 24 for use in an in vivo method for diagnosing a disease characterised by presenting cells with differential expression of an antigen.

29. Functionalised magnetic nanoparticle or pharmaceutical composition for use according to claim 28, wherein the disease characterised by presenting cells with differential expression of an antigen is selected from cancer, a cardiovascular disease and a bacterial infection.

30. Functionalised magnetic nanoparticle for use according to claim 29 wherein the cancer is selected from the group consisting of pancreatic cancer, lung cancer, colon cancer, bladder cancer, endometrial cancer, kidney cancer, breast cancer, stomach cancer, non-Hodgkin's lymphoma, thyroid cancer, soft-tissue sarcoma, metastatic breast cancer, ovarian cancer, prostate cancer and rectal cancer.

31. Use of the functionalised magnetic nanoparticle according to any of claims 1 to 15 and 20 or the pharmaceutical composition according to claim 24 as a contrast agent for imaging.