WO2015135597A1

WO2015135597A1 - Uses and methods for delivery to the nucleus

Info

Publication number: WO2015135597A1
Application number: PCT/EP2014/059710
Authority: WO
Inventors: Lianbing ZHANG; Le Li; Mato Knez
Original assignee: Cic Nanogune - Asociación Centro De Investigación Cooperativa En Nanociencias
Priority date: 2014-03-12
Filing date: 2014-05-13
Publication date: 2015-09-17

Abstract

The present invention relates to uses of ferritin nanoparticles for delivering agents to the nucleus. The present invention also relates to methods for delivering agents to the nucleus using ferritin nanoparticles. Furthermore, the present invention also relates to ferritin nanoparticles for uses in therapy.

Description

USES AND METHODS FOR DELIVERY TO THE NUCLEUS

FIELD OF THE INVENTION The present invention relates to the use of a ferritin nanoparticle for the delivery of agents to the nucleus as well as to methods for the delivery of agents to the nucleus. The invention also relates to agent-loaded ferritin nanoparticles and their applications. Further the invention relates to a composition comprising, separately, empty ferritin nanoparticles and an anti-cancer agent and its applications in the treatment of cancer.

BACKGROUND OF THE INVENTION

Efficient subcellular-targeted delivery is critical for administering drugs with distinct nucleocytoplasmic therapeutic targets and for gene therapy. Although viral vectors are employed in most worldwide clinical gene therapy trials, inherent viral risks still restrict their clinical use. Non-viral vectors are less efficient and usually need multiple modifications to become biocompatible, enhance cellular uptake and facilitate nuclear entry. The emergence and application of nanotechnology in drug delivery is still a very challenging prospect in both pharmaceutics and disease therapy. Currently, many novel nanoparticles have been widely used to improve the delivery of various drugs using the following approaches: (i) reducing the particle size to nanometer size, which will increase both the surface area of the carrier and the rate of dissolution, and (ii) improving bioavailability by increasing the adsorption level of insoluble compounds, which may result in reducing the amount of dose required and the side effects. In addition, various nanoparticle ligands play an important role in developing target drug delivery. These techniques can greatly improve the delivery capacities of drugs and their utilization rate, including the low water-solubility of drugs, are aimed at target delivery of drugs in a cell or tissue specific manner; and allow transcytosis of drugs across epithelial and endothelial barriers. One of such nanoparticles is the ferritin nanocage or nanoparticle. Ferritin is a conserved protein for iron storage which ubiquitously exists in animals, plants and bacteria. The molecular structure of ferritin is very unusual, and differs from most proteins in nature; it has a large cavity whose diameter can reach 8-10 nm, and its protein shell is composed of 24 subunits of H-chain and/or L-chain type, which can be dissociated and re-assembled into the original protein shell by regulating the pH medium. The large cavity within the ferritin shell has enough space to store small molecular compounds, including heavy metals, anticancer drugs and peptides. WO 2003/094849 A2 describes a ferritin fusion protein, in which L- or H-chains are fused with a protein or peptide. The protein or peptide may be inside the nanoparticle when fused to the C-terminus of ferritin, and/or on the outer surface when fused to the N-terminus. The proteins or peptides include antibodies, antibody fragments, enzymes, fragments, peptidoglycans and peptides, provided that they do not interfere with the polymeric self-assembly of ferritin. These fusion proteins may be used for therapeutic applications, including the targeted delivery of encapsulated drugs to target tissues. However, this document does not provide further details.

Thompson et al. (2002, J Cell Sci 115:2165-77) describes the regulation and mechanisms of H-ferritin translocation to the nucleus in SW1088 astrocytoma cells. Results shown further demonstrate that L-ferritin does not translocate to the nucleus. However, the assay conditions included permeabilisation of the cell membrane with digitonin at 4°C for 5 min followed by the addition of ferritin, which are far remote from standard physiological conditions.

Li et al. (2010, Proc Natl Acad Sci USA 107:3505-10) identifies human transferrin receptor- 1 (TfR-1) as an important receptor for H-ferritin with little or no binding to L- ferritin. The bound H-ferritin internalizes and enters endosomes, being also able to sort to lysosomes. However, no localisation to the nucleus was observed.

WO 2008/048288 A2 describes the targeting of ferromagnetic iron oxide inside ferritin nanoparticles to tumour cells. The targeting is mediated by an RGD-loop fused to the N- terminus of H-ferritin, which is known to bind to ανβ3 integrin. The nanoparticles were found to localise on the surface of C32 melanoma cells after 30 min incubation at 37°C although the intracellular localisation was not evaluated. Zhen et al. (2013, ACS Nano 7:4830-7) describes ferritin nanoparticles that are used for the targeted delivery of doxorubicin to U87MG human glioblastoma cells. The targeting is mediated by an RGD-loop fused to the N-terminus of H-ferritin, which is known to bind to ανβ3 integrin, an integrin overexpressed in U87MG cells. The incubation conditions were 37°C for at least 30 min. The nanoparticles were shown to internalise the cells, with doxorubicin being detected in the nucleus after 2 h of incubation. No intracellular localisation of the ferritin nanoparticles was provided.

Ji et al. (2012, J Proteomics 75:3145-57) describes ferritin and apoferritin nanoparticles containing cisplatin that are based on pig pancreas ferritin, as well as a method for loading the ferritin nanoparticle with cisplatin in alkaline medium. The composition in L- and H-chains is not provided. Both the ferritin and apoferritin nanoparticles were able to internalise into GCC and HeLa cells after being incubated at 37°C for 8 h. Ferritin nanoparticles are shown to localise in the cytoplasm after 8 h of incubation. However, no nuclear localisation was observed.

In view of this, there is still a need in the art for a system for the rapid and efficient delivery of agents to the cell nucleus.

BRIEF DESCRIPTION OF THE INVENTION

It has been found that ferritin nanoparticles consisting of ferritin H-chains only (H- ferritin) or a mixture of ferritin H-chains and L-chains translocate into the nucleus upon being internalised by the cell, but the translocation does not occur when the ferritin nanoparticles consist of L-chains only. The translocation occurs regardless of whether the ferritin nanoparticles are empty, i.e. apoferritin, or loaded with an agent. Therefore the inventive aspects and objects mentioned below fall within the scope of the present patent application.

In an aspect, the invention relates to the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for delivering the agent to the nucleus of a target cell. In a particular embodiment, the use of said ferritin nanoparticle for delivering the agent to the nucleus of a target cell is performed in vitro. In another particular embodiment, the use of said ferritin nanoparticle for delivering the agent to the nucleus of a target cell is performed in vivo.

The invention also relates to a method for delivering an agent to the nucleus of a target cell which comprises contacting a ferritin nanoparticle comprising at least one ferritin H-chain and an agent with a target cell under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell. In a particular embodiment, the method for delivering the agent to the nucleus of a target cell is performed in vitro. In another particular embodiment, the method for delivering the agent to the nucleus of a target cell is performed in vivo. The invention also relates to a ferritin nanoparticle comprising at least one ferritin H- chain and an agent for use in the treatment of a disease, wherein said agent is an agent that is indicated for the treatment of said disease.

The invention also relates to the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for the manufacture of a pharmaceutical composition the treatment of a disease, wherein said agent is an agent that is indicated for the treatment of said disease.

The invention also relates to a method for the treatment of a disease which comprises administering to a subject in need thereof a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent is an agent that is indicated for the treatment of said disease. The invention also relates to a ferritin nanoparticle comprising at least one ferritin H- chain and an agent, wherein the agent comprises an imaging agent, for use in in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

The invention also relates to the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for in vivo delivering the agent to the nucleus of a target cell, or for in vivo visualizing the nucleus of a target cell.

The invention also relates to the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, in the manufacture of a pharmaceutical composition for in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

The invention also relates to an in vivo method for delivering an agent to the nucleus of a target cell in a subject, or for in vivo visualizing the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle comprising at least one ferritin H- chain and an agent with a target cell, wherein the agent comprises an imaging agent.

The invention also relates to an in vitro use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for delivering the agent to the nucleus of a target cell, or for visualising the nucleus of a target cell.

The invention also relates to an in vitro method for delivering an agent to the nucleus of a target cell, or for visualising the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle comprising at least one ferritin H-chain and said agent with a target cell, wherein the agent comprises an imaging agent, under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell. The invention also relates to a novel ferritin nanoparticle comprising at least one ferritin H-chain and an agent with the proviso that:

the agent is not a metal,

- if the agent is doxorubicin, then the ferritin nanoparticle does not consist of 24 ferritin H-chains,

if the agent is cisplatin, then the ferritin nanoparticle does not consists of pig ferritin chains, and

if the ferritin nanoparticle consists of human ferritin chains and comprises two agents, wherein one of the agents is cisplatin, then the other agent is not the monoclonal antibody Epl specific to the human melanoma antigen chondroitin sulfate proteoglycan 4 (CSPG4).

The invention also relates to said novel ferritin nanoparticle for use in the treatment of a disease, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

The invention also relates to the use of said novel ferritin nanoparticle for the manufacture of a pharmaceutical composition the treatment of a disease, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

The invention also relates to a method for the treatment of a disease which comprises administering to a subject in need thereof said novel ferritin nanoparticle, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

The invention also relates to said novel ferritin nanoparticle, wherein the agent comprises a non-metal imaging agent, for use in in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell. The invention also relates to the use of said novel ferritin nanoparticle, wherein the agent comprises a non-metal imaging agent, for in vivo delivering the agent to the nucleus of a target cell, or for in vivo visualizing the nucleus of a target cell. The invention also relates to the use of said novel ferritin nanoparticle, wherein the agent comprises a non-metal imaging agent, in the manufacture of a pharmaceutical composition for in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell. The invention also relates to an in vivo method for delivering an agent to the nucleus of a target cell in a subject, or for in vivo visualizing the nucleus of a target cell, the method comprising contacting said novel ferritin nanoparticle, wherein the agent comprises a non-metal imaging agent. The invention also relates to an in vitro use of said novel ferritin nanoparticle, wherein the agent comprises a non-metal imaging agent for delivering the agent to the nucleus of a target cell, or for visualising the nucleus of a target cell.

The invention also relates to an in vitro method for delivering an agent to the nucleus of a target cell, or for visualising the nucleus of a target cell, the method comprising contacting said novel ferritin nanoparticle, wherein the agent comprises a non-metal imaging agent, with a target cell, wherein the agent comprises a non-metal imaging agent, under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell.

The invention also relates to a pharmaceutical composition, selected from: a) a pharmaceutical composition comprising a therapeutically effective amount of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein said agent is an agent indicated for the treatment of a disease, together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle; and b) a pharmaceutical composition comprising a therapeutically effective amount of said novel ferritin nanoparticle, wherein the agent is an agent indicated for the treatment of a disease, together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle.

The invention also relates to a composition comprising, separately, (i) a ferritin nanoparticle comprising at least one ferritin H-chain and (ii) an anti-cancer agent. The invention also relates to a pharmaceutical composition comprising, separately, (i) a pharmaceutical composition comprising a ferritin nanoparticle comprising at least one ferritin H-chain together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle, and (ii) a pharmaceutical composition comprising an anti-cancer agent together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle.

The invention also relates to any of the above said compositions comprising, separately, (i) a ferritin nanoparticle comprising at least one ferritin H-chain and (ii) an anti-cancer agent, for use in the treatment of cancer. The invention also relates to the use of any of the above said compositions in the manufacture of a medicament comprising separately a (i) a pharmaceutical composition comprising a ferritin nanoparticle comprising at least one ferritin H chain and (ii) a pharmaceutically composition comprising an anti-cancer agent. The invention also relates to a method for the treatment of cancer which comprises administering to a subject in need thereof of any of the above said compositions comprising, separately, (i) a ferritin nanoparticle comprising at least one ferritin H-chain and (ii) an anti-cancer agent. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Nucleocytoplasmic distribution and nuclear translocation of cagelike apoferritins. (a) The cells were treated with 200 μΜ DFO for 48 h to remove the endogenous ferritin (+DFO). The untreated cells were stained as the normal cells (norm. Cell). H-ferritin was detected with a rabbit anti-H-ferritin antibody together with the FITC-conjugated anti-rabbit antibody (green). The nuclei were stained with propidium iodide (red). The detection with the HRP-conjugated secondary antibody only served as negative control (N. Contr.). Scale bars: 18.75 μιη (N. Contr. and +DFO) and 34.13 μιη (nornxCell). (b) Western blot detection of H-ferritin and biotinylated H- and L- apoferritin in the cytoplasmic and nuclear fractions. The fractions from the untreated cells loaded as control (Contr.). (c) Western blot detection of biotinylated human H- apoferritin in cytoplasmic and nuclear fractions of Caco-2 cells treated with 50 μg/ml biotinylated H-apoferritin for various durations. 20 μg total protein from the nuclear fractions was loaded for the SDS-PAGE. (d) Western blot detection of biotinylated human L- and H-apoferritin in cytoplasmic and nuclear fractions of Caco-2 cells treated with 50 μg/ml biotinylated apoferritin. The biotin-L/H is ferritin formed from L- and H- chains in 1 : 1 ratio. 25 μg/ml of each biotin-L and biotin-H were supplemented for the co-treatment. Respectively, 7 μg and 20 μg total protein from the cytoplasmic and nuclear fractions were loaded for the SDS-PAGE. (e) The schematic presentation of the distinct nucleocytoplasmic delivery locations of the L- and H-ferritin.

Figure 2: Detection of H-ferritin and biotin. (a) Detection of H-ferritin and biotin.

(a), detection of H-ferritin in cytolasmic fractions with an anti-H-ferritin antibody, (b), detection of biotin in the cytoplasmic and nuclear fractions with the HRP-StrepTactin. The H-biotin (red) indicated the biotinylated H-ferritin loaded as a control. The deferoxamine (DFO) pretreatment (200 μΜ for 48h) was followed with the treatments with 50 μg/ml free biotin, biotinylated L- (L-biotin) and H-ferritin (H-biotin). The fractions from the untreated cells loaded as control (Contr.). 7 μg and 20 μg from each cytoplasmic and nuclear fraction, respectively, were loaded on the gel. Figure 3: Encapsulation of Dox in H-ferritin. 1 mg/ml disassembled H-ferritin was added to 20 mg/ml Dox solution with the pH value of 9. After the purification, the UV- vis absorption of H-Dox was compared with the spectra of H-ferritin and free Dox. Dox has a characteristic absorption peak at 485 nm.

Figure 4. Rapid nuclear translocation of the anti-cancer drug and effects of the surface modification. Live cell imaging of (a) Hep G2 and (b) Caco-2 cells treated with 10 μg/ml Dox encapsulated in human H-apo ferritin (H-Dox), 20 μg/ml free Dox or Dox encapsulated in liposomes (Lipo-Dox). Scale bar: 20 μιη. (c) Western blot detection of streptavidin in cytoplasmic and nuclear fractions. The fractions from untreated cells were loaded as control (Contr.). (d) Immunofluorescence detection of streptavidin (green). Streptavidin was conjugated with human L- and H-ferritin (L-strep and H-strep) or 10 nm gold nanoparticles (Au-strep), with or without DFO pretreatment (± DFO, 200 μΜ for 48 h). Subsequently, the cytoplasmic and nuclear fractions of the cells were collected after 45 min treatments with free streptavidin or the streptavidin conjugates. The nucleus staining was accomplished with propidium iodide (red). Scale bars of the triple images are 37.5 μιη, 21.7 μιη, 19.37 μιη, 18.75 μιη, 18.75 μιη and 23.65 μιη in downward order.

Figure 5: Nuclear translocation of H-Dox. (a) Caco-2 and (b) Hep G2 cells were treated with 10 μg/ml or 20 μg/ml free Dox, or 10 μg/ml Dox encapsulated in human H- apoferritin (H-Dox) or 50 μg/ml Dox encapsulated in liposomes (Lipo-Dox). The cytoplasmic Dox that was not intercalated with DNA was washed away during the fixation. The fixed cells were subjects for laser scanning confocal microscopy. The nuclei were stained with DAPI (blue). Scale bar: 20 μιη. Figure 6: Nuclear translocation of ferritin lowers the dose of Dox. The cells were treated for 15 minutes or 2 hours with 10 μg/ml and 20 μg/ml free Dox or 10 μg/ml Dox encapsulated in human L-apo ferritin. The fixed cells were subjects for laser scanning confocal microscopy. The nuclei were stained with DAPI. Scale bar: 20 μιη. Figure 7: Detection of streptavidin. (a), detection of streptavidin in the cytoplasmic fractions with an anti-streptavidin antibody. The monomer of streptavidin is 15 kDa. β- actin (42 kDa) as the loading control on the same membrane. The membrane was stripped before the actin detection. 7 μg protein of each fraction was loaded on the gel. The fractions from the untreated cells loaded as control (Contr.). (b), detection of streptavidin in the nuclear fractions. The cells were treated with deferoxamine (DFO, 200 μΜ for 48h), followed with the treatments with 50 μg/ml free streptavidin (strep), streptavidin conjugated L- (L-strep) and H-ferritin (H-strep). The fractions from the untreated cells loaded as control (Contr.). 20 μg protein of each fraction was loaded on the gel. The H-strep (red) indicated the streptavidin conjugated H-ferritin loaded as a control. Figure 8: Nuclear translocation of streptavidin. The cells were treated with DFO (± DFO, 200 μΜ for 48 h). Subsequently, the cells were treated for 45 min with streptavidin conjugated H-ferritin (+H- ferritin). Streptavidin (green) is immunhistochemically detected. The nucleus staining was accomplished with propidium iodide (red). Scale bars: 75 μιη.

Figure 9: Cellular distribution of L-ferritin. The detection only with the HRP- conjugated secondary antibody served as the negative control (N. Contr.). The untreated cells were stained as the normal cells (norm. Cell). L-ferritin was detected also in cells treated with the streptavidin conjugated L-ferritin (+L-strep). L-ferritin was detected with a rabbit anti-L- ferritin antibody together with the FITC-conjugated anti-rabbit antibody. The nuclei were stained with propidium iodide. Scale bars: 18.75 μιη.

Figure 10: Confocal microscope top-down and side profile views analysis of cells treated with the streptavidin conjugated H-ferritin. Top down view (left panel middle), X-Z view (left panel below) and Y-Z view (left panel right); Y-Z view (right panel) shows the localizations of streptavidin (upper), nucleus (middle) and merge image (below). Scale bars: 10.72 μιη.

Figure 11. Bypassing the multidrug resistance and synergistic effects, (a), cellular multidrug resistance level after treatment with 5 μg/ml Dox (either as free Dox or encapsulated in liposome or ferritin) for 24 h. The multidrug resistance activity of untreated cells was set as 0 and used as control (contr.). (b), the cellular iron quantification of the iron challenged cells treated for 24 h with free Dox and Dox encapsulated in liposomes (Lipo-Dox) or human H-apoferritin (Dox-huFH). The Dox concentration was 5 μg/ml for all treatments. The iron challenge was performed with addition of 50 μΜ ferric citrate for 24 h. The change of the intracellular iron level is based on the comparison of the intracellular iron with untreated cells, (c), Western blot detection of the transferrin receptor (TfR) and ferroportin in the cell membrane fractions and (d) HIF-Ι in the nuclear fraction after the treatment indicated in (b). The detection was carried out on the identical membrane. Na⁺/K⁺ATPase and TATA served as the loading control for membrane and nuclear proteins, respectively. The results in (a) and (b) are represented by the mean ± SD. * : P < 0.01 ; * * : P < 0.001.

Figure 12: Cell viability upon the treatments. The cells was treated with the deferoxamine (DFO, 300 μΜ for 48h), followed by treatments with 100 μg/ml free streptavidin (strep), streptavidin conjugated gold nanoparticles (Au-strep), L- (L-strep) and H-ferritin (H-strep). The viability of the untreated cells serves as the control with 100% viability (Contr.). The results are shown as value ± SD.

DETAILED DESCRIPTION OF THE INVENTION The inventors of the present invention have identified that ferritin nanoparticles consisting of ferritin H-chains only (H-ferritin) or a mixture of ferritin H-chains and L- chains translocate into the nucleus upon being internalised by the cell, but the translocation does not occur when the ferritin nanoparticles consist of L-chains only. The cellular uptake and the subsequent nuclear translocation of supplemented H-chain ferritin nanoparticles follows a surprisingly rapid process, which brings an unmatchable advantage for delivery of encapsulated agents efficiently and with a reduced dose. As shown in Example 1, ferritin chains are detected in the nucleus as early as 15 min from exposure to the cell. The translocation occurs regardless of whether the ferritin nanoparticles are empty, i.e. apoferritin, or loaded with agents either in their internal cavity or on their outer surface. This system has the additional advantage of achieving nuclear translocation of ferritin in its intact cage-structure thereby escaping cellular mechanisms such as the activation of the cellular multidrug resistance. The invention will be described in detail below.

Definitions

The meaning of some terms and expressions as they are used in the present description are indicated below to aid in understanding.

The term "ferritin", as used herein, refers to a highly conserved protein that keeps iron in a bioavailable form and protects DNA from oxidative and UV-induced damages. It consists of a mineral core of hydrated ferric oxide, and a 24-subunit protein shell that encloses the former and assures its solubility in an aqueous environment. The demineralised form of ferritin is apo ferritin. Both ferritin and apoferritin are composed of ferritin H- and L-chains or subunits that are highly conserved and nevertheless genetically different. The ferritin H- and L-chains spontaneously assemble in a 24- subunit globular protein with an internal or inner cavity.

The term "apoferritin", as used herein, refers to the demineralised form of ferritin, i.e. the ferritin protein lacking ferric oxide.

The term "ferritin H-chain" or "ferritin H-subunit" or "H-chain" or "H-subunit" or "heavy chain", as used herein, refers to the ferritin heavy chain also known as FTH1, FTHL6, and cell proliferation- inducing gene 15 protein. The human ferritin H-chain is identified in the UniProt database under accession number P02794 on 12^th February 2014.

The term "ferritin L-chain" or "ferritin L-subunit" or "L-chain" or "L-subunit" or "light chain", as used herein, refers to the ferritin light chain also known as FTL. The human ferritin L-chain is identified in the UniProt database under accession number P02792 on 12^th February 2014. The term "ferritin nanoparticle" or "ferritin nanocage", as used herein, refers to the 24- subunit protein shell of ferritin or apoferritin, indistinctly. The self-assembled 24-mer ferritin nanoparticle comprises an inner cavity and an outer surface that is formed by the 24 ferritin chains. The ferritin nanoparticle has a flexible H-chain to L-chain ratio. Thus, the ferritin nanoparticle may be composed of H-chains only, L-chains only, or a variable mixture of H-chains and L-chains. Typical dimensions of the nanoparticles include an internal diameter of 7.6 nm, although it is able to reach 8-10 nm, and an outer diameter of 12 nm. Distributed around the sphere, there are 14 small channels, each 3-4 A in diameter, perforating the protein shell and providing size selectivity for ions or molecules to enter the interior or inner cavity. For the uses of the present invention, the ferritin nanoparticle comprises at least one H-ferritin chain.

Uses for delivering agents to the nucleus Thus, in an aspect, the present invention relates to the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for delivering the agent to the nucleus of a target cell. In a particular embodiment, the use of said ferritin nanoparticle for delivering the agent to the nucleus of a target cell is performed in vitro. In another particular embodiment, the use of said ferritin nanoparticle for delivering the agent to the nucleus of a target cell is performed in vivo.

In another aspect, the invention relates to a method for delivering an agent to the nucleus of a target cell which comprises contacting a ferritin nanoparticle comprising at least one ferritin H-chain and an agent with a target cell under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell. In a particular embodiment, the method for delivering the agent to the nucleus of a target cell by using said ferritin nanoparticle is performed in vitro. In another particular embodiment, the method for delivering the agent to the nucleus of a target cell by using said ferritin nanoparticle is performed in vivo. The ferritin nanoparticle comprising at least one ferritin H-chain and an agent can be used in in vitro applications for delivering the agent to the nucleus of a target cell, for example, in in vitro imaging applications, in genetic engineering applications, in screening of therapeutic agents on a target cell, etc., as well as in in vivo applications for delivering the agent to the nucleus of a target cell, for example, in diagnosis and in therapeutic applications.

A. Nanoparticle As described in the context of the definitions of the invention, the ferritin nanoparticle is formed with the self-assembly of 24 ferritin subunits, which creates an inner cavity of about 7.6 nm diameter, although it is able to reach about 8-10 nm, and an outer surface of about 12 nm diameter. The composition in ferritin chains of the ferritin nanoparticle can vary, generally, from all ferritin H-chains, i.e. 24 H-chains, to all ferritin L-chains, i.e. 24 L-chains. However, the present invention only contemplates the use of ferritin nanoparticles comprising at least 1 ferritin H-chain for the delivery of agents to the nucleus of a target cell. This includes the use of ferritin nanoparticles consisting of 1 H-chain and 23 L-chains, of 2 H-chains and 22 L-chains, of 3 H-chains and 21 L-chains, of 4 H-chains and 20 L- chains, of 5 H-chains and 19 L-chains, of 6 H-chains and 18 L-chains, of 7 H-chains and 17 L-chains, of 8 H-chains and 16 L-chains, of 9 H-chains and 15 L-chains, of 10 H-chains and 14 L-chains, of 11 H-chains and 13 L-chains, of 12 H-chains and 12 L- chains, of 13 H-chains and 11 L-chains, of 14 H-chains and 10 L-chains, of 15 H-chains and 9 L-chains, of 16 H-chains and 8 L-chains, of 17 H-chains and 7 L-chains, of 18 H- chains and 6 L-chains, of 19 H-chains and 5 L-chains, of 20 H-chains and 4 L-chains, of 21 H-chains and 3 L-chains, of 22 H-chains and 2 L-chains, of 23 H-chains and 1 L- chains, and of 24 H-chains. In a particular embodiment, the ferritin nanoparticle for use according to the invention consists of ferritin H-chains. It will be understood by the person skilled in the art that a ferritin nanoparticle consisting only of H-chains is a ferritin nanoparticle composed of (or consisting of) 24 ferritin H-chains. This type of ferritin nanoparticle is also known as H-ferritin.

B. Ferritin

The particulars of "ferritin", "H-chain" and "L-chain" have been mentioned in the "Definitions" section and are incorporated herein by reference.

The biological origin or source of the ferritin chains which are present in the ferritin nanoparticle for use according to the invention can vary broadly, e.g. it can be from any mammal, such as primates and humans, domestic animals, farm animals, and zoo, sports, or pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on. However, in a particular embodiment, the ferritin is human ferritin. In a particular embodiment, the ferritin H-chain is human ferritin H-chain or a functional variant or fragment thereof, preferably, human ferritin H-chain. In another particular embodiment, the ferritin L-chain is human ferritin L-chain or a functional variant or fragment thereof, preferably human ferritin L-chain. As used herein, the term "functional variant of ferritin" refers to a peptide or protein resulting from the addition, deletion or substitution of one or more amino acid residues from the sequence of said ferritin chain, wherein said ferritin is selected from the group consisting of the ferritin H-chain and the ferritin L-chain, and that substantially maintains its capacity to assemble in the 24-mer protein with an inner cavity. Functional variants of a ferritin chain according to the invention include polypeptides showing a sequence identity with the sequence of said ferritin chain of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% that retain capacity to assemble in the 24-mer protein with an inner cavity. Likewise, functional variants of a ferritin chain according to the invention will preferably have a capacity to assemble in the 24-mer protein with an inner cavity of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the capacity of said ferritin chain to assemble in the 24-mer protein with an inner cavity. The degree of identity between two proteins or peptides can be determined by using computer-implemented algorithms and methods that are widely known by those skilled in the art. By illustrative, the identity between two amino acid sequences is determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al, J. Mol. Biol, 1990, 215:403-410). The reassembly of the ferritin nanoparticles can be evaluated with native gel electrophoresis or size-exclusion chromatography (Anal Biochem. 1987, 166(2):235-45).

As used herein, the term "functional fragment of ferritin" also refers to a peptide or protein with identical sequence as that of said ferritin chain, wherein said ferritin is selected from the group consisting of the ferritin H-chain and the ferritin L-chain, with a deletion of at least 1 amino acid from its N-terminus, and/or a deletion of at least 1 amino acid from its C-terminus, and that substantially maintains its capacity to assemble in the 24-mer protein with an inner cavity. Functional variants of ferritin considered within the context of this invention include polypeptides the sequence of which is derived from the sequences mentioned above by the deletion of at least 1 amino acid, 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, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids from its N-terminus, and/or a deletion of at least 1 amino acid, 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, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, at least 100 amino acids from its C-terminus that substantially maintains the capacity to assemble in the 24-mer protein with an inner cavity. Functional fragments of ferritin will preferably have a capacity to assemble in the 24-mer protein with an inner cavity of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the capacity of said ferritin chain to assemble in the 24-mer protein with an inner cavity. The reassembly of the ferritin nanoparticles can be evaluated as mentioned above with native gel electrophoresis or size-exclusion chromatography. The capacity to assemble in the 24-mer protein with an inner cavity is considered to be substantially maintained if the ferritin H-chain variant retains at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 98%, at least 99%, or 100% of the capacity to assemble in the 24-mer protein with an inner cavity of the wild type ferritin H-chain.

In another particular embodiment, the ferritin is apoferritin. In a preferred embodiment, the apoferritin is human apoferritin.

C. Agent

As the person skilled in the art will recognise, the structural characteristics of the ferritin nanoparticles comprising at least one ferritin H-chain and their ability to rapidly translocate into the cell nucleus (see Example 1) may be employed for the nuclear delivery of an agent.

As used herein, within the context of nuclear delivery, the term "agent" refers to an entity suitable for exerting an activity in the cell nucleus. Agents may be useful, among other applications, for analytical, therapeutic and imaging applications, and research applications, in various forms. Agents suitable for the present invention comprise, or consist of, without limitation, proteins, peptides, hormones, nucleic acids, small molecules, metals, etc., products that can act as reagents, imaging agents, drugs, etc. The agents can be delivered to the cell nucleus where they exert their function or, alternatively, they can specifically target the ferritin particle to the target cell. Thus, in an embodiment, the agent of the ferritin nanoparticle comprises, or consists of, a protein, a peptide, a nucleic acid, a small molecule, or a metal. In a particular embodiment, the agent comprises, or consists of, a protein. As used herein, the term "protein" refers to a macromolecule consisting of one or more chains of amino acid residues. Proteins are responsible for carrying out a diverse set of cellular functions based on their ability to bind other molecules specifically and tightly. Proteins can bind to other proteins as well as to small molecule substrates. Protein-protein interactions can regulate enzymatic activity, control progression through the cell cycle, and allow the assembly of large protein complexes that carry out many closely related reactions with a common biological function. Non limitative examples of proteins include:

- enzymes, such as DNA/RNA processing enzymes, etc.;

- proteins involved in cell signalling and ligand binding, such as protein hormones, growth factors, cytokines, antibodies, DNA/RNA binding proteins, etc.; and

structural proteins, such as collagen, elastin, keratin, actin, tubulin, etc.

Thus, in another particular embodiment the agent is a protein selected from the group consisting of an enzyme, an antibody, a DNA binding protein, and an RNA binding protein. In a preferred embodiment, the protein is an enzyme. As used herein, the term "enzyme" refers to a biological macromolecule that functions as a highly selective catalyst, greatly accelerating both the rate and specificity of a metabolic reaction for which it is specific. Enzymes may be proteins or catalytic RNA molecules. In the context of the present invention, it is most appropriate that the enzyme is capable of exerting its function in the cell nucleus. For this reason, DNA and/or RNA processing enzymes are particularly preferred. Non- limitative examples of DNA and/or RNA processing enzymes include nucleases, such as restriction enzymes, homing endonucleases (HEGs) and zinc-finger nucleases (ZNFs); recombinases, such as Cre, MerCreMer and variants thereof; integrases (Buchholz F, 2009, Curr Opinion Biotech 20: 1-7), etc. In another preferred embodiment, the protein is an antibody. 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 (Ausubel et al, ed., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY 1987-2001; Sambrook, et al, Molecular Cloning: A Laboratory Manual, 4^th Edition, Cold Spring Harbor, NY, 2012; Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY, 1989; Colligan, et al, ed., Current Protocols in Immunology, John Wiley and Sons, Inc., NY 1994-2001; Colligan et al, Current Protocols in Protein Science, John Wiley and Sons, NY, NY, 1997-2001; Kohler et al, Nature 256:495-497, 1975; US 4, 816, 567, Queen et al, Proc. Natl. Acad. Sci. 86: 10029-10033, 1989). 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), etc. In another preferred embodiment, the protein is a DNA binding protein or an RNA binding protein. Examples of DNA binding proteins or RNA binding proteins include, without limitation, transcription factors, SR proteins and the ADAR protein.

In another particular embodiment, the 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, to bind to a target, or peptide aptamers.

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

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. In another preferred embodiment, the peptide is an aptamer.

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).

As used herein, the term "aptamer" or "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 macro molecular 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.

In another particular embodiment, the agent comprises, or consists of, a nucleic acid. As used herein, the term "nucleic acid" refers to oligomeric or polymeric molecules made from monomers known as nucleotides. Nucleic acids include DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). Each nucleotide has three components: a 5-carbon sugar, a phosphate group, and a nitrogenous base. If the sugar is deoxyribose, the nucleic acid is DNA, and if the sugar is ribose, the nucleic acid is RNA.

Thus, in a preferred embodiment, the nucleic acid comprises, or consists of, DNA. DNA molecules suitable for use in the context of the present invention include, without limitation, a plasmid and a DNA oligonucleotide. In a more preferred embodiment, the DNA is a plasmid. In another more preferred embodiment, the DNA is a DNA oligonucleotide. The term "plasmid", as used herein, refers to a DNA molecule that is physically separate from, and can replicate independently of, chromosomal DNA within a cell. Plasmids are widely used as vectors for the integration of DNA constructs in the cell genome, and applications include the generation of disease models and gene therapy. Techniques for the preparation and use of the various plasmids are well known in the art (Sambrook, et al. cited supra).

The term "DNA oligonucleotide", as used herein, refers to a short chain of nucleotides linked by phosphodiester bonds. The DNA oligonucleotide will comprise at least 2 nucleotides, at least 3 nucleotides, at least 4 nucleotides, at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, at least 20 nucleotides, at least 30 nucleotides, at least 40 nucleotides, or at least 50 nucleotides. DNA oligonucleotides have a wide range of bio techno logical and therapeutic applications. Examples of DNA oligonucleotides include, without limitation, DNA aptamers and antisense DNA oligonucleotides. 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). The term "antisense DNA oligonucleotide" or "antisense DNA", as used herein, refers to a single strand DNA that is complementary to a chosen sequence. Antisense DNA can be used to target a specific, complementary (coding or non-coding) RNA, and the resulting DNA/RNA hybrid can be degraded by the enzyme RNase H. Antisense DNA oligonucleotides are particularly useful for gene knockdowns in vertebrates to study altered gene expression and gene function.

In another preferred embodiment, the nucleic acid comprises, or consists of, RNA. RNA molecules suitable for use in the context of the present invention include, without limitation, an RNA antisense oligonucleotide, a small hairpin RNA (shRNA), a small interfering RNA (siRNA), and a microRNA (miRNA). In a more preferred embodiment, the RNA is an RNA antisense oligonucleotide. In another more preferred embodiment, the RNA is a shRNA. In another more preferred embodiment, the RNA is a siRNA. In another more preferred embodiment, the RNA is a miRNA.

The term "antisense RNA oligonucleotide" or "antisense RNA", as used herein, refers to a single strand RNA that is complementary to a chosen sequence. Antisense RNA can be used to prevent protein translation of certain messenger RNA strands by binding to them. Antisense RNA oligonucleotides are particularly useful for gene knockdowns in vertebrates to study altered gene expression and gene function.

The term "small hairpin RNA" or "short hairpin RNA" or "shRNA", as used herein, refers to a sequence of RNA that makes a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. Due to the ability of shRNA to provide specific, long-lasting, gene silencing there has been great interest in using shRNA for gene therapy applications. The term "small interfering RNA" or "short interfering RNA" or "silencing RNA" or "siRNA", as used herein, refers to a class of double-stranded RNA molecules of about 20-25 nucleotides in length. siRNA plays many roles, but it is most notable in the RNAi pathway, where it interferes with the expression of specific genes with complementary nucleotide sequence.

The term "micro R A" or "miRNA", as used herein, refers to a small non-coding RNA molecule of about 20-25 nucleotides in length, which functions in transcriptional and post-transcriptional regulation of gene expression. miRNAs function via base-pairing with complementary sequences within mRNA molecules, usually resulting in gene silencing via translational repression or target degradation. RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression, typically by causing the destruction of specific mRNA molecules. Given the ability to knock down, in essence, any gene of interest, RNAi via shRNA, siRNAs and miRNA is widely used in both basic and applied biology, in applications such as gene knockdown, functional genomics, and therapy, including cancer and antiviral therapy. Non- limitative examples of targets suitable for this therapeutic approach include the vascular endothelial growth factor (VEGF) and kinesin spindle protein (KSP).

In another particular embodiment, the agent comprises, or consists of, a small molecule. As used herein, the term "small molecule" refers to a low molecular weight [i.e., equal to or less than (<) 900 Daltons] organic compound that may help regulate a biological process, with a size on the order of 10 ⁹ m. Most drugs are small molecules. The upper molecular weight limit for a small molecule is approximately 900 Daltons, which allows for the possibility to rapidly diffuse across cell membranes so that they can reach intracellular sites of action. In addition, this molecular weight cutoff is a necessary but insufficient condition for oral bioavailavility. A lower molecular weight cutoff of 500 Daltons has been recommended for small molecule drug development candidates based on the observation that clinical attrition rates are significantly reduced if the molecular weight is kept below this 500 Dalton limit. Small molecules can have a variety of biological functions, serving as cell signaling molecules, as drugs in medicine, as pesticides in farming, and in many other roles. These compounds can be natural, such as secondary metabolites, for example, alkaloids, glycosides, lipids, nonribosolmal peptides (e.g., actinomycin-D), phenazines, natural phenols (including flavonoids), polyketide, terpenes (including steroids), tetrapyrroles, etc.), or artificial (such as some drugs, i.e., chemically derived, man-made compounds developed to treat a wide range of diseases); they may have a beneficial effect against a disease (such as drugs) or may be detrimental (such as teratogens and carcinogens). Biopolymers such as nucleic acids, proteins, and polysaccharides (such as starch or cellulose) are not small molecules, although their constituent monomers, ribo- or deoxyribo-nucleotides, amino acids, and monosaccharides, respectively, are often considered small molecules. Very small oligomers are also usually considered small molecules, such as dinucleotides, peptides such as the antioxidant glutathione, and disaccharides such as sucrose. In a particular embodiment, the small molecule is a molecule that binds to a specific biopolymer, such as a protein or a nucleic acid, and acts as an effector, altering the activity or function of the biopolymer. Further, small molecules may also be used as research tools to probe biological function as well as leads in the development of new therapeutic agents. Some can inhibit a specific function of a multifunctional protein or disrupt protein-protein interactions.

In another particular embodiment, the agent comprises, or consists of, a metal. As used herein, the term "metal" refers to an element that readily forms positive ions (cations) and has metallic bonds. Metals are sometimes described as a lattice of positive ions surrounded by a cloud of delocalized electrons. The metals are one of the three groups of elements as distinguished by their ionization and bonding properties, along with the metalloids and nonmetals. On the periodic table, a diagonal line drawn from boron (B) to polonium (Po) separates the metals from the nonmetals. Most elements on this line are metalloids, sometimes called semi-metals; elements to the lower left are metals; elements to the upper right are nonmetals. Examples of agents comprising, or consisting of, metals include, without limitation, a metal alloy, a metallic complex, a metal oxide and a metal nanoparticle.

The agent of the ferritin nanoparticle comprising at least one ferritin H-chain, in an embodiment, comprises, or consists of, a hormone, an imaging agent, or a drug. As used herein, the term "hormone" refers to a chemical messenger that transports a signal from one cell to another, i.e., with endocrine functions in living organisms. The term "hormone" includes both "peptide hormone" and "non-peptide hormones". Illustrative, non- limitative examples of peptide hormones include angiotensin II, basic fibroblast growth factor-2, parathyroid hormone-related protein, prolactin, adrenocorticotropic hormone (ACTH), growth hormone, vasopressin, oxytocin, atrial- natriuretic peptide (ANP), atrial natriuretic factor (ANF), glucagon, insulin, somatostatin, cholecystokinin, gastrin, leptin, etc. Illustrative, non-limitative examples of non-peptide hormones include an androgen, an oestrogen, Cortisol, progesterone, vitamin A, T3, and T4. In the context of the present invention, hormones that bind to nuclear receptors are particularly advantageous since they regulate the expression of specific genes. In a particular embodiment, the hormone is an oestrogen.

In another particular embodiment, the agent comprises, or consists of, an imaging agent. As used herein, the term "imaging agent" refers to a chemical compound that is designed to allow the localization 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.

In another particular embodiment, the agent comprises, or consists of, a drug. As used herein, 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. A preferred class of drugs are drugs that intervene at the nuclear level in the cell. 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 and an anthracycline.

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, carboplatin 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 5-fluorouracil (5FU), floxuridine (FUDR), cytosine arabinoside (cytarabine), 6-azauracil (6-AU), etc.; or

antifolates, such as methotrexate, pemetrexed, proguanil, pyrimethamine, trimethoprim, etc. 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 R A 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.

Illustrative, non- limitative examples of anthracyclines include daunorubicin, doxorubicin, epirubicin, idarubicin, valrubicin, mitoxantrone, etc.

In a preferred embodiment, the drug is an anthracycline, preferably, doxorubicin. In another preferred embodiment, the drug is bevacizumab, capecitabine, cisplatin, cyclophosphamide, epirubicin, 5-fluorouracil, folinic acid, methotrexate, or oxaliplatin.

The structure of the ferritin nanoparticle comprising at least one ferritin H-chain enables the delivery of an agent wherein said agent is either within the inner cavity or on the outer surface or both. For the incorporation of agents in the cavity it is required that the size of the agents does not interfere with the polymeric self-assembly of ferritin chains, and that the ferritin is demineralised so that the inner cavity is substantially empty, i.e., apoferritin.

The inner cavity is considered to be substantially empty if content of ferric oxide is less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.1%, less than 0.01%, less than 0.001%, or less than 0.0001% of the ferric oxide content of the ferritin nanoparticle when is fully mineralised. The agent can be located within the ferritin nanoparticle comprising at least one ferritin H-chain within the inner cavity, or on the outer surface, or, even, a part of the agent may be located within the ferritin nanoparticle inner cavity whereas another part of the agent may be located on the outer surface of the ferritin nanoparticle.

Thus, in a particular embodiment, the agent is substantially located within the nanoparticle inner cavity, i.e., all agent molecules, or a very substantial amount thereof, are contained within the ferritin nanoparticle inner cavity. Thus, according to the invention, a portion of at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the agent is located within the ferritin nanoparticle inner cavity. In another particular embodiment, the agent is substantially located on the outer surface of the nanoparticle, i.e., all agent molecules, or a very substantial amount thereof, is on the ferritin nanoparticle outer surface. Thus, according to the invention, a portion of at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the agent is located on the outer surface of the ferritin nanoparticle.

In another particular embodiment, a portion of the agent molecules is located within the ferritin nanoparticle inner cavity whereas another portion of the agent molecules is located on the outer surface of the ferritin nanoparticle.

In an embodiment, the invention contemplates the nuclear delivery of just one agent. In another embodiment, the invention also contemplates the nuclear delivery of a second (or further) agent(s). Thus in another particular embodiment, the ferritin nanoparticle further comprises, in addition to a first agent, a second agent, wherein said second agent is an agent that is different from said first agent. Thus, by illustrative, if the first agent is, for example, a protein (e.g., "Protein P"), the second agent can be a protein other than the protein of the first agent (i.e., other than "Protein P"), or any other agent such as a peptide, an hormone, a nucleic acid, an imaging agent, a metal or a drug; similarly, if the first agent is an hormone (e.g., "Hormone H"), the second agent can be an hormone other than the hormone of the first agent (i.e., other than "Hormone H"), or another agent such as a protein, a peptide, a nucleic acid, an imaging agent, a metal or a drug.

In the particular embodiment where the ferritin nanoparticle for use according to the invention comprises two or more agents, the person skilled in the art will easily understand that what has been mentioned in connection with the agent is also fully applicable to the second agent that can be present in the ferritin nanoparticle as mentioned above, provided that the second agent is other than the first agent. Thus, in a preferred embodiment, the second agent for use in the instant invention comprises, or consists of, without limitation, a protein, a peptide, a nucleic acid, a small molecule, or a metal that can be delivered to the cell nucleus where they exert their function or, alternatively, that can specifically target the ferritin particle to the target cell. In another preferred embodiment, the second agent for use in the instant invention comprises, or consists of, a hormone, an imaging agent, or a drug. The particulars of said agents have been previously disclosed in connection with the agent (i.e., the first agent) (Section C) and are herein incorporated by reference. The skilled person in the art will also easily understand that if the ferritin nanoparticle comprises a third (or further) agent, said third (or further) agent will have the same nature as the first agent and will be subjected to the same provision as that of the second agent (i.e., they will be different from the first and second agents). It is also contemplated that the second (or further) agent(s) is substantially located within the inner cavity or on the outer surface of the ferritin nanoparticle comprising at least one ferritin H-chain, regardless of the location of the first agent within, or on, the ferritin nanoparticle. The invention also contemplates that a portion of the second (or further) agent(s) is located within the nanoparticle inner cavity whereas another portion of the second (or further) agent(s) is located on the outer surface of the ferritin nanoparticle, irrespective of the location of the first agent within, or on, the nanoparticle. Thus, as an illustrative example, the first agent may be substantially located on the outer surface of the ferritin nanoparticle and the second (or further) agent(s) may be substantially located within the inner cavity of the ferritin nanoparticle; or the first agent may be substantially located within the inner cavity of the ferritin nanoparticle and the second (or further) agent(s) may be substantially located on the outer surface of the ferritin nanoparticle; or the first and the second (or further) agent(s) may be substantially located within the inner cavity of the ferritin nanoparticle; or the first and the second (or further) agent(s) may be substantially located on the outer surface of the ferritin nanoparticle. Thus, in a particular embodiment, the first agent and the second agent are substantially located within the nanoparticle inner cavity. In another particular embodiment, the first agent and the second agent are substantially located on the outer surface of the nanoparticle. In another particular embodiment, the first agent is substantially located within the nanoparticle inner cavity and the second agent is substantially located on the outer surface of the nanoparticle. In another particular embodiment, the first agent is substantially located on the outer surface of the nanoparticle and the second agent is substantially located within the nanoparticle inner cavity. In another particular embodiment, a portion of the first agent, as well as a portion of the second agent, is located within the nanoparticle inner cavity whereas another portion of the first agent, as well as a portion of the second agent, is located on the outer surface of the nanoparticle.

D. Production of agent-loaded ferritin nanoparticles The ferritin nanoparticle comprising at least one ferritin H-chain and an agent for use according to the present invention can be obtained by any method that allows the location of the agent within the ferritin nanoparticle inner cavity or on the outer surface of the ferritin nanoparticle, for example, any method comprising the addition of the agent to the ferritin nanoparticle, including encapsulation or generation of fusion proteins and protein conjugates. It will be appreciated that agents may be delivered within the inner cavity of the ferritin nanoparticle, either via encapsulation in the nanoparticle or covalently linked to the C- terminus of a ferritin chain. Agents may additionally be covalently coupled to a ferritin chain to be delivered on the outer surface of the ferritin nanoparticle. The linkage of agents may occur on the ferritin H-chain or on the ferritin L-chain if it was present. Thus, agents are generally incorporated in ferritin nanoparticle either by means of encapsulation or by means of covalent coupling.

Several methods have been developed in the art to encapsulate agents in the inner cavity of ferritin nanoparticles. Briefly, in a particular embodiment, a process is based on the disassociation of the ferritin subunits at an acidic pH, for instance about 2.0, or basic pH, for instance about 13, in the presence of the agent to be encapsulated, and the subsequent re-assembly of the ferritin subunits on return to neutral pH, thereby trapping the agent within the inner cavity so formed (Simsek & Kilic, 2005, J Magnetism Magnet Materials 293:509-13; Yang et al, 2007, Chem Commun (33):3453-5; Ji et al, 2012, cited supra). An alternative method for encapsulation of smaller molecules or metal ions comprises diffusion of the molecules or ions through the pores at the intersections of the protein subunits of the assembled ferritin as previously described by Wong & Mann (Wong & Mann, 1996, Adv. Mater. 8 (11): 928-32).

Ferritin nanoparticles comprising at least one ferritin H-chain and two or more agents encapsulated within the nanoparticle inner cavity for use according to the present invention can be obtained as mentioned above by introducing the necessary modifications.

Alternatively, agents may be incorporated in the ferritin nanoparticle by means of covalent coupling. This can be achieved by way of chemical conjugation of the agent with a ferritin chain, usually by crosslinking. As used herein, the term "conjugate" refers to the product resulting from the chemical joining of a first agent and one ferritin chain comprised in the ferritin nanoparticle by a non-peptidic, chemical bond, normally by means of a linker. This chemical bond is typically achieved by crosslinking. As used herein, the term "crosslinking", "conjugation" or "chemical conjugation" refers to the process of chemically joining two or more components by a covalent, non-peptidic bond. There are a number of different conjugation chemistries specific for the different functional groups available in the art, and these need to be selected according to the nature of the agent to be conjugated to the ferritin chain, i.e. a protein, a peptide, a hormone, a nucleic acid, an imaging agent, or a drug. This is common knowledge to the skilled person.

Methods of conjugation are well known in the art and are based on the use of linking reagents. Linking or crosslinking reagents contain at least two reactive groups, which target common functional groups, such as primary amines, sulfhydryls, aldehydes, carboxyls, hydroxyls, azides and so on, on the molecule to be conjugated. The crosslinking agents differ in chemical specificity, spacer arm length, spacer arm composition, spacer arm cleavability, and structure. For example, protein conjugation can be carried out directly or through a linker moiety, through one or more non- functional groups on the agent and/or the ferritin chain, such as amine, carboxyl, phenyl, thiol or hydroxyl groups. Conventional linkers can be used, such as diisiocyanates, diisothiocyanates, bis(hydroxysuccinimide) esters, carbodiimides, maleimide-hydroxysuccinimide esters, glutaraldehyde and the like, or hydrazines and hydrazides, such as 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH).

A simple method for making a protein:protein conjugate is to mix the two proteins in the presence of glutaraldehyde to form the conjugate. The initial Schiff base linkages can be stabilized, e.g., by borohydride reduction to secondary amines. A diisothiocyanate or a carbodiimide can be used in place of glutaraldehyde.

More selective linkage can be achieved by using a heterobifunctional linker such as a maleimide-hydroxysuccinimide ester. Reaction of the latter with an enzyme will derivatise amine groups on, e.g., an enzyme, and the derivative can then be reacted with, e.g., an antibody fragment with free sulfhydryl groups.

It is advantageous to link the agent to a site on the ferritin chain remote from the site of self-assembly. Non-limiting examples of methods to accomplish this include linkage to cleaved interchain sulfhydryl groups, as noted above. Another method involves reacting, e.g. a peptide whose carbohydrate portion has been oxidized with a ferritin chain which has at least one free amine function. This results in an initial Schiff base (imine) linkage, which is preferably stabilised by reduction to a secondary amine, e.g., by borohydride reduction, to form the final conjugate. See also the discussion above of the methods described in U.S. Pat. No. 5,772,981 and hydrazine and hydrazide linkages.

The linker may be a "cleavable linker", i.e., a linker that facilitates release of the first agent and/or second agents in the cell nucleus. For example, an acid-labile linker, peptidase-sensitive linker, dimethyl linker or disulfide-containing linker may be used.

When the agent comprises a protein or a peptide, it is also possible to covalently link said agent to at least one ferritin chain by means of a peptidic bond, resulting in the agent being fused to the ferritin chain. As used herein, the term "fusion protein" or "chimeric protein" refers to the expression product of a gene encoding a (polypeptide comprising the agent linked by a peptide (amide) bond to an at least one ferritin chain. The fusion of at least these two components is such that one is allowed to move with respect to the other, and that it does not cause a substantial loss in the activity of one or all the components.

The person skilled in the art will understand that it may be desirable that the fusion protein further comprises a flexible peptide that binds the agent and the at least one ferritin chain. The flexible peptide will permit the movement of one component with respect to the other.

Therefore, in a particular embodiment, the fusion protein comprising an agent fused to at least one ferritin chain further comprises a flexible peptide.

As used herein, the term "flexible peptide", "spacer peptide" or "linker peptide" refers to a peptide that covalently binds the agent and the at least one ferritin chain, which is not part of neither the agent nor the at least one ferritin chain, and which allows movement of one component with respect to the other, without causing a substantial detrimental effect on the function of either component. In a preferred embodiment, said flexible peptide binds the gent and the at least one ferritin chain substantially without causing a detrimental effect on the function of neither the agent and the at least one ferritin chain. It is not necessary that the agent and the at least one ferritin chain are arranged in that order and, in this case, the invention contemplates fusion proteins in which the agent is located at the amino -terminal position relative to the at least one ferritin chain, and wherein the agent is located at the carboxyl-terminal position relative to the at least one ferritin chain. In the particular embodiment where the ferritin nanoparticle comprises two or more agents of peptidic or protein nature, the present invention contemplates that the first agent and the second (or further) agent(s) are fused to the at least one ferritin chain that can be the same ferritin chain or different ferritin chains.

The flexible peptide comprises at least one amino acid, at least two amino acids, at least three amino acids, at least four amino acids, at least five amino acids, at least six amino acids, at least seven amino acids, at least eight amino acids, at least nine amino acids, the least 10 amino acids, at least 12 amino acids, at least 14 amino acids, at least 16 amino acids, at least 18 amino acids, at least 20 amino acids, at least 25 amino acids, at least 30 amino acids, at least 35 amino acids, at least 40 amino acids, the least 45 amino acids, at least 50 amino acids, at least 60 amino acids, at least 70 amino acids, at least 80 amino acids, at least 90 amino acids, or about 100 amino acids.

Flexible peptides suitable for use in the present invention include those which have been previously described as suitable for linking two polypeptide domains that allow said polypeptide domains to substantially retain their native structure and activity, such as those disclosed in WO2009150284. In a preferred embodiment, the peptide linker is made up mainly of the residues glycine, serine and/or proline. Peptide linkers suitable for use in the invention include peptides comprising the sequences (GlySer)_n, (Gly_mSer)_n or (Ser_mGly)_n, wherein m is 1 to 6, particularly 1 to 4, and typically 2 to 4, n is 1 to 30 or 1 to 10 and typically 1 to 4 and, optionally, comprise some residues of glutamic acid (Glu) or lysine (Lys) distributed along the sequence to enhance solubility (see, for example, WO 96/06641). In a particular embodiment, the agent is fused to the ferritin chain through a spacer.

In another particular embodiment, the agent is fused to at least one ferritin chain. In this particular embodiment, it is possible that the agent is fused to at least one ferritin H- chain or to at least one ferritin L-chain if the ferritin nanoparticle comprises both H- chain(s) and L-chain(s). The skilled person in the art will understand that what is mentioned for just one agent can be applied to two or more agents by introducing the necessary modifications.

Thus, in a particular embodiment, the agent is fused to at least one ferritin chain. In another particular embodiment, the agent is conjugated to at least one ferritin chain.

The ferritin nanoparticle of the invention may comprise one, two or more agents. In the particular embodiment where the ferritin nanoparticle comprises a first agent and a second (or further) agent(s), said first agent and said second (or further) agent(s) may be fused or conjugated to at least one ferritin chain. In another particular embodiment, the first agent is fused to at least one ferritin chain. In another particular embodiment, the first agent is conjugated to at least one ferritin chain. In another particular embodiment, the second agent is fused to at least one ferritin chain. In another particular embodiment, the second agent is conjugated to at least one ferritin chain.

E. In vivo and in vitro uses It will be instantly appreciated by the person skilled in the art that the nuclear delivery of an agent may be used for in vivo applications as well as for in vitro applications. Thus, the ferritin nanoparticle comprising at least one H-ferritin and an agent may be used in vivo or in vitro for the delivery of the agent to a target cell. E. l In vivo uses In vivo uses of the ferritin nanoparticle comprising at least one ferritin H-chain and an agent include, without limitation, uses in therapeutic applications as well as uses in diagnostic applications, for example, by means of imaging, and uses in therapeutic applications.

E.l . l Therapy

In a particular embodiment, the agent is an agent that is indicated (i.e., for use or suitable) for treating a disease, and, therefore, the ferritin nanoparticle comprising at least one ferritin H-chain and said agent can be used in the treatment of said disease for which the agent is indicated by in vivo delivering of said agent to the nucleus of a target cell.

Therefore, in a particular embodiment, the invention contemplates a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for use in the treatment of a disease, wherein said agent is an agent that is indicated for the treatment of said disease, or alternatively expressed as the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for the manufacture of a pharmaceutical composition the treatment of a disease, wherein said agent is an agent that is indicated for the treatment of said disease.

Further, according to this particular embodiment, the invention also contemplates an in vivo method for the treatment of a disease which comprises administering to a subject in need thereof a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent is an agent that is indicated for the treatment of said disease.

According to the invention, the agent indicated for the treatment of the disease is delivered to the nucleus of the target cell by means of the ferritin nanoparticle comprising at least one ferritin H-chain which further comprises said agent. The terms "ferritin nanoparticle", "ferritin H-chain" and "agent" and their particulars have been previously described in Sections A, B, C and E. l, and said terms and their particulars are incorporated herein by reference. As used herein, the term "target cell" refers to the particular cell that internalises the ferritin nanoparticle. Upon being internalised, the ferritin nanoparticle translocates in intact form to the nucleus, in a cytological mechanism that is both efficient and rapid. Any cell can potentially be targeted by ferritin nanoparticles provided that they contain ferritin receptors, for example, the transferrin receptor 1 (TfRl), the transferrin receptor 2, etc. In a particular embodiment, the target cell contains a ferritin receptor. In another particular embodiment, the target cell contains the transferrin receptor 1 (TfRl).

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.

According to the present invention, the ferritin nanoparticle comprising at least a ferritin H-chain and an agent for use in 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. The person skilled in the art will immediately know which agents are indicated for the treatment of a particular disease. Nearly all agents that are indicated for treating a disease may be comprised in the ferritin nanoparticle comprising at least a ferritin H- chain, although those agents that exert their activity in the cell nucleus are particularly preferred. Therefore, the ferritin nanoparticle comprising at least a ferritin H-chain and an agent can be used in the treatment of virtually any disease capable of benefiting from the treatment with the appropriate therapeutic agent.

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 in detail in Section C and are incorporated here by reference.

In the context of the radionuclides useful for radiotherapy, alpha-emitting, beta-emitting and gamma-emitting radionuclides are particularly useful. Agents 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, agents 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 agent, 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 agent is selected from the group consisting of a radionuclide and a drug. In a preferred embodiment, the agent is a radionuclide. In another preferred embodiment, the agent is a drug. In a more preferred embodiment, the agent is an antitumoral drug.

In another particular embodiment, said disease capable of benefiting from the treatment with the appropriate agent (i.e., an agent indicated for the treatment of said disease) comprised in the ferritin nanoparticle comprising at least a ferritin H-chain and said agent is selected from the group consisting of a tumour disease and a non-tumour disease. In a preferred embodiment, the disease is a tumour disease or cancer. The term cancer has been described previously and the term and its particulars are herein incorporated by reference.

It is common knowledge in the art which agents 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

Cancer type Drugs

Cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin,

Breast cancer

bevacizumab

Stomach cancer Epirubicin, cisplatin, 5-fluorouracil, capecitabine

Bladder cancer Methotrexate, vincristine, doxorubicin, cisplatin

Lung cancer Cyclophosphamide, doxorubicin, vincristine, bevacizumab

Colorectal

5-Fluorouracil, folinic acid, oxaliplatin

cancer

Brain cancer Bevacizumab These therapeutic applications will comprise the administration of a therapeutically effective amount of the ferritin nanoparticle comprising at least a ferritin H-chain and the appropriate agent. The term "therapeutically effective amount", as used herein, refers to the amount of said ferritin nanoparticle comprising at least a ferritin H-chain and an agent, wherein said agent is an agent useful for the treatment of a disease for which said agent is indicated, which is required to achieve an appreciable cure or killing of cells of said disease. The ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein said agent is indicated for treating a disease is particularly efficient in the treatment of said disease because it undergoes a rapid translocation with its load into the nucleus upon being internalised by the target cell. Thus, in another particular embodiment, the ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein said agent is indicated for treating a disease, is internalised by the target cell and translocates into the nucleus.

Example 4 of the present invention shows that the ferritin nanoparticles disclosed herein achieve nuclear delivery of the agent whilst avoiding the exposure of the agent to the various cellular mechanisms. Nuclear translocation without cytoplasmic exposure is another advantage associated with ferritin as the drug carrier which enables the drug to circumvent several antineoplastic resistance mechanisms, such as, drug efflux and enzymatic inactivation. It will be understood by the person skilled in the art that, in order to fully exploit potential of the ferritin nanoparticle comprising at least one ferritin H-chain and an agent of circumventing the cellular resistance mechanisms, it would be necessary that the agent is located within the nanoparticles' cavity. For the incorporation of an agent in the cavity it is required that the size of the agent does not interfere with the polymeric self-assembly of ferritin chains, and that the ferritin is demineralised so that the inner cavity is substantially empty, i.e. apoferritin. Details about the location of the agent in the ferritin nanoparticles have been described in detail previously and are incorporated here by reference. Thus, in a particular embodiment, the agent is substantially located in the ferritin inner cavity. In another particular embodiment, the agent is substantially located on the ferritin outer surface.

In a particular embodiment, the ferritin nanoparticle comprising at least one ferritin H- chain and an agent further comprises, in addition to said agent (i.e. the first agent), a second (or further) agent, which is different to the first agent, wherein said first and second agents, independently each other, are suitable for the treatment of a disease. In a particular embodiment, said second (or further) agent may also be indicated for treating the same disease as the first agent. This is particularly advantageous when the two (or more) therapeutic agents have additive or synergistic activity that complements and that do not adversely affect each other. This particular ferritin nanoparticle can be used in the treatment of virtually any disease capable of benefiting from the treatment with one or two or more agents. Thus in a preferred embodiment, the second (or further) agent is indicated for treating said disease. Alternatively, the second (or further) agent may be indicated for treating a different disease, or may not be indicated for treating any disease. In another preferred embodiment, the second (or further) agent(s) is not indicated for treating the same disease as that susceptible of being treated with the first agent. For the administration to a subject in need thereof of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein said agent is an agent indicated for the treatment of said disease, said nanoparticle will be formulated in a suitable pharmaceutical composition. The particulars of said pharmaceutical composition will be discussed below.

E.1.2 Diagnosis

In another particular embodiment, the agent comprises an imaging agent, and, therefore, the ferritin nanoparticle comprising at least one ferritin H-chain and said agent can be used in in vivo delivering said agent to the nucleus of a target cell, or for visualizing the nucleus of a target cell, thus finding application in diagnostics, especially in in vivo diagnosis by imaging techniques. Therefore, in a particular embodiment, the invention contemplates the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for in vivo delivering the agent to the nucleus of a target cell, or for in vivo visualizing the nucleus of a target cell; or, alternatively, as a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for use in in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell; or, alternatively, the use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, in the manufacture of a pharmaceutical composition for in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

Further, according to this particular embodiment, the invention also contemplates an in vivo method for delivering an agent to the nucleus of a target cell in a subject, or for in vivo visualizing the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle comprising at least one ferritin H-chain and an agent with a target cell, wherein the agent comprises an imaging agent. The terms "ferritin nanoparticle", "ferritin H-chain", "agent", "imaging agent", "target cells" and their particulars have been previously described in Sections A, B and C, and said terms and their particulars are incorporated herein by reference.

The person skilled in the art will immediately know the suitable imaging techniques which can be used taking into account the imaging agent which is present in the ferritin nanoparticle comprising at least a ferritin H-chain and said imaging agent.

Έ.2 Ιη vitro uses In vitro uses of the ferritin nanoparticle comprising at least one H-ferritin and an agent include, without limitation, uses in imaging applications, for the specific visualization and/or detection of the cell nucleus of a target cell; uses in genetic engineering applications, such as, for example, to genetically modify a cell in vitro, wherein the target cell is an established cell line as well as a somatic cell, a germ cell and a stem cell isolated from a subject; and uses for the screening of therapeutic agents on a target cell. In a particular embodiment, the invention contemplates the in vitro use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for delivering the agent to the nucleus of a target cell, or for visualising the nucleus of a target cell; or an in vitro method for delivering an agent to the nucleus of a target cell, or for visualising the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle comprising at least one ferritin H-chain and said agent with a target cell, wherein the agent comprises an imaging agent, under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell. According to the invention, the imaging agent is delivered to the nucleus of the target cell by means of the ferritin nanoparticle comprising at least one ferritin H-chain which further comprises said agent in order to visualise the nucleus of a target cell.

The terms "ferritin nanoparticle", "ferritin H-chain", "agent", "imaging agent" and "target cell" and their particulars have been previously described in Sections A, B, C and E.1 , and said terms and their particulars are incorporated herein by reference.

As used herein, the term "conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell" includes the conditions in which the ferritin nanoparticle is able to be internalized by the target cell and translocates to the nucleus. These conditions include temperature, humidity, concentration of gases and culture medium and contact time. Typically, the temperature, humidity, concentration of gases and culture medium will depend on the type of target cell and will be the suitable conditions for culturing said cells. The standard temperature for culturing cells, specially human cells, is 37°C. The term "contact time", as used herein, refers to the time that runs from the moment when the ferritin nanoparticle is contacted with the target cell until the moment when said ferritin nanoparticle is internalized by the target cell and translocates to the nucleus. The contact time is of at least 1 min, at least 2 min, at least 3 min, at least 4 min, at least 5 min, at least 10 min, at least 15 min, at least 20 min, at least 25 min, at least 25 min, at least 30 min, at least 35 min, at least 40 min, at least 45 min, at least 50 min, at least 55 min, at least 1 h, at least 2 h, at least 3 h, at least 4 h, at least 5 h, or more.

The target cell may be isolated or in the form of a culture. Cells can be grown either in suspension or adherent cultures. Some cells naturally live in suspension, without being attached to a surface, such as cells that exist in the bloodstream. There are also cell lines that have been modified to be able to survive in suspension cultures so they can be grown to a higher density than adherent conditions would allow. Adherent cells require a surface, such as tissue culture plastic or microcarrier, which may be coated with extracellular matrix (such as collagen and/or laminin) components to increase adhesion properties and provide other signals needed for growth and differentiation. Most cells derived from solid tissues are adherent. Another type of adherent culture is an organotypic culture, which involves growing cells in a three-dimensional (3D) environment as opposed to two-dimensional (2D) culture dishes. This 3D culture system is biochemically and physiologically more similar to in vivo tissue. The suitable conditions for culturing cells depend on the target cells and are generally known by the skilled person in the art.

The delivery of the imaging agent to the nucleus of a target cell and/or the nucleus of the target cell to which the imaging agent has been delivered, can be visualized by any suitable imaging technique taking into account the nature of the imaging agent, such as, for example, fluorescence microscopy, PET, SPECT, gamma imaging, etc.

In another particular embodiment, the invention contemplates the in vitro use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent in genetic engineering applications, such as, for example to genetically modify a target cell in vitro. The target cell may be an established cell line, or, alternatively, a somatic cell, a germ cell or a stem cell isolated from a subject. The target cell can be in vitro genetically modified and, then, if desired, implanted into a subject in order to correct any deficiency. In another particular embodiment, the invention contemplates the in vitro use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for the screening of therapeutic agents on a target cell.

F. Ferritin nanoparticles of the invention

In another aspect, the invention relates to a ferritin nanoparticle, hereinafter referred to as the "ferritin nanoparticle of the invention", comprising at least one ferritin H-chain and an agent with the proviso that:

the agent is not a metal,

The terms "ferritin nanoparticle", "ferritin H-chain", "agent" and their particulars have been previously described in detail in Sections A, B and C, and the terms and their particulars are herein incorporated by reference.

In a particular embodiment of the ferritin nanoparticle of the invention, the agent comprises a protein, a peptide, a nucleic acid, or a small molecule. In another particular embodiment of the ferritin nanoparticle of the invention, the agent comprises a hormone, a non-metal imaging agent or a drug. The ferritin nanoparticle of the invention can be used, among others, in analytical, therapeutic and imaging applications, as well as in research applications.

As it is evident for any skilled person in the art, the ferritin nanoparticle of the invention can be used in any of the in vitro and in vivo uses of the ferritin nanoparticle comprising at least one ferritin H-chain and an agent previously disclosed based on the delivery of an agent to the nucleus of a target cell.

Thus, the invention also contemplates a ferritin nanoparticle of the invention for use in the treatment of a disease, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease, or the use of a ferritin nanoparticle of the invention for the manufacture of a pharmaceutical composition for the treatment of a disease, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

The invention also contemplates a method for the treatment of a disease which comprises administering to a subject in need thereof a ferritin nanoparticle of the invention, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

Preferred particulars embodiments of the above mentioned uses and methods include an agent wherein the agent comprises a radionuclide or comprises a drug; or wherein the agent comprises a drug and the drug is selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline, such as, for example, bevacizumab, capecitabine, cisplatin, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, folinic acid, methotrexate, or oxaliplatin; and/or wherein the disease is a cancer.

Further, the invention also contemplates, a ferritin nanoparticle of the invention, wherein the agent comprises a non-metal imaging agent, for use in in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell; or the use of a ferritin nanoparticle of the invention, wherein the agent comprises a non-metal imaging agent, for in vivo delivering the agent to the nucleus of a target cell, or for in vivo visualizing the nucleus of a target cell; or the use a ferritin nanoparticle of the invention, wherein the agent comprises a non-metal imaging agent, in the manufacture of a pharmaceutical composition for in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

The invention also contemplates an in vivo method for delivering an agent to the nucleus of a target cell in a subject, or for in vivo visualizing the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle of the invention, wherein the agent comprises a non-metal imaging agent.

Preferred particulars embodiments of the above mentioned uses and methods include a ferritin nanoparticle of the invention which comprises, in addition to a non-metal imaging agent, a second agent, wherein said second agent is other than said non-metal imaging agent; or wherein the target cell is a tumour cell.

The invention also contemplates an in vitro use of a ferritin nanoparticle of the invention, wherein the agent comprises a non-metal imaging agent, for delivering the agent to the nucleus of a target cell, or for visualising the nucleus of a target cell.

Further, the invention also contemplates an in vitro method for delivering an agent to the nucleus of a target cell, or for visualising the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle of the invention, wherein the agent comprises a non-metal imaging agent, with a target cell, wherein the agent comprises a non-metal imaging agent, under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell.

G. Pharmaceutical compositions

For the administration to a subject in need thereof of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein said agent is an agent indicated for the treatment of a disease, or a ferritin nanoparticle of the invention wherein the agent is an agent indicated for the treatment of a disease, said nanoparticles will be formulated in suitable pharmaceutical compositions. Thus, in another aspect, the present invention relates to a pharmaceutical composition, selected from: a pharmaceutical composition comprising a therapeutically effective amount of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein said agent is an agent indicated for the treatment of a disease, together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle [sometimes referred to as "pharmaceutical composition a)"]; and a pharmaceutical composition comprising a therapeutically effective amount of a ferritin nanoparticle of the invention [i.e., a ferritin nanoparticle comprising at least one ferritin H-chain and an agent with the proviso that (i) the agent is not a metal, (ii) if the agent is doxorubicin, then the ferritin nanoparticle does not consists of 24 ferritin H-chains, (iii) if the agent is cisplatin, then the ferritin nanoparticle does not consists of pig ferritin chains, and (iv) if the ferritin nanoparticle consists of human ferritin chains and comprises two agents, wherein one of the agents is cisplatin, then the other agent is not the monoclonal antibody Epl specific to human melanoma antigen CSPG4], wherein the agent is an agent indicated for the treatment of a disease, together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle [sometimes referred to as "pharmaceutical composition b) of the invention"].

The pharmaceutical composition provided by the present invention can be used for treating a disease for which the agent is indicated, such as, for example, for curing or killing cells of a disease susceptible of benefiting from the treatment with the therapeutic agent that has its activity in the nucleus. The term "pharmaceutically acceptable carrier", as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter- ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Supplementary active compounds can also be incorporated into the pharmaceutical composition provided by the present invention. Thus, in a particular embodiment, the pharmaceutical composition provided by the present invention may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a chemotherapeutic agent, a cytokine, an analgesic agent, or an immunosuppressive agent. The effective amount of such other active agents depends, among other things, on the amount of the therapeutically loaded ferritin nanoparticles which are present in the pharmaceutical composition, the nature and severity of the disease being treated, the patient, etc. In an embodiment, the ferritin nanoparticles comprising at least one ferritin H-chain and an agent, wherein said agent is an agent indicated for the treatment of a disease, or the ferritin nanoparticle of the invention, are formulated with carriers that will protect said products against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. 4,522, 811.

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 intratumoral 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 sterilization. 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 (therapeutic ferritin 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 ferritin nanoparticles 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 that has its activity in the nucleus, specially, for treating tumour diseases or cancers.

H. Compositions of the invention and combination therapy

The inventors have also shown that the ferritin nanoparticles comprising at least one ferritin H-chain can suppress the upregulation of HIF-Ι in tumour cells (see Example 6). This effect occurs independently of the agent contained in the ferritin nanoparticle and hence is the result of the ferritin nanoparticle that has been translocated into the nucleus. Since the up-regulation of HIF-Ι is a common phenomenon in tumour cells, the present invention also contemplates the use of ferritin nanoparticles comprising at least one ferritin H-chain as an additive or synergistic compound for chemotherapy of tumours, i.e. in combination therapy. Thus, in another aspect, the invention relates to a composition, hereinafter referred to as the "composition of the invention", comprising, separately, (i) a ferritin nanoparticle comprising at least one ferritin H chain and (ii) an anti-cancer agent.

The terms "ferritin nanoparticle", "ferritin H-chain", and "cancer", and their particulars have been described previously in detail in Sections A, B and E, and are incorporated herein by reference.

The term "anti-cancer agent" or "anti-tumoural agent", as used herein, refers to an agent that is useful in the treatment of cancer. Anti-cancer agents include radionuclides and drugs such as those radionuclides and drugs previously described which are also incorporated here by reference. Additionally, other anti-cancer agents include, without limitation, the following agents:

angiogenesis inhibitors, such as angio statin, endostatin, fumagillin, genistein, minocycline and staurosporin;

- DNA synthesis inhibitors, such as aminopterin, ganciclovir and hydroxyurea; enzyme inhibitors, such as S(+)-camptothecin, curcumin, 2-Imino-l-imidazoli- dineacetic acid (Cyclocreatine), hispidin, formestane, and mevinolin; microtubule inhibitors, such as colchicine and dolastatin 15; and

other anti-tumour agents, such as 17-(allylamino)-17-demethoxygeldanamycin, apigenin, cimetidine, luteinizing hormone-releasing hormone, and pifithrin-a. The agents indicated for the treatment of a particular cancer are common knowledge in the art. By way of an illustrative example, a relation of type of cancers and drugs that are indicated for the treatment of said types of cancers is given in Table 1. Thus, in a particular embodiment, a ferritin nanoparticle comprising at least one ferritin H chain is used in combination with an anti-cancer agent for the treatment of a cancer. In a particular embodiment, said anti-cancer agent is any of the anti-cancer agents mentioned herein. In a more particular embodiment, said anti-cancer agent is any of the anti-cancer agents mentioned in Table 1. In a specific particular embodiment, said anticancer agent is doxorubicin. In another preferred embodiment, the drug is bevacizumab, capecitabine, cisplatin, cyclophosphamide, epirubicin, 5-fluorouracil, folinic acid, methotrexate, or oxaliplatin.

For the administration of components (i) and (ii) of the composition of the invention to the subject in need thereof, each component, will be separately formulated in separate, suitable pharmaceutical compositions for their simultaneous, or sequential, administration to the subject in need thereof.

Thus, it is also contemplated within the scope of the present invention a pharmaceutical composition, hereinafter referred to as "pharmaceutical composition [2] of the invention", comprising, separately, (i) a pharmaceutical composition comprising a ferritin nanoparticle comprising at least one ferritin H-chain together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle, and (ii) a pharmaceutical composition comprising an anti-cancer agent together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle.

The composition of the invention as well as the pharmaceutical composition [2] of the invention, is therefore useful in a combination therapy for the treatment of cancer. As used herein, the term "combination therapy" or "polytherapy" refers to the use of more than one agent or therapy for the treatment of a single disease, wherein the disease is a cancer or tumour. One major benefit of combination therapies is that they reduce development of drug resistance, since a tumour is less likely to have resistance to multiple drugs simultaneously. Combination therapies may comprise at least two different therapies, as is the case of the present invention.

Each pharmaceutical formulation will be specific to the corresponding component of the composition of the invention. For example, the ferritin nanoparticle comprising at least one ferritin H-chain will be in a suitable pharmaceutical composition, and the anti- cancer agent will be in another suitable pharmaceutical composition. To that end, said components will be formulated together with the appropriate pharmaceutically acceptable excipients, carriers or vehicles, such as it has been mentioned previously in Section G, and it is incorporated herein by reference. The components of the composition of the invention may be administered simultaneously or sequentially (i.e., separated by time), and may be administered in either order, in the same or different pharmaceutical forms for administration, and/or by the same or different routes. The person skilled in the art will appreciate that the composition of the invention is particularly useful for therapeutic applications, specifically for the treatment of cancer.

Thus, in another aspect, the invention relates to a composition of the invention, or a pharmaceutical composition [2] of the invention, for use in the treatment of cancer, or to the use of a composition of the invention, in the manufacture of a medicament comprising separately a (i) a pharmaceutical composition comprising a ferritin nanoparticle comprising at least one ferritin H chain, and (ii) a pharmaceutically composition comprising an anti-cancer agent. In another aspect, the invention relates to a method for the treatment of cancer which comprises administering to a subject in need thereof a therapeutically effective amount of a composition of the invention, or of a pharmaceutical composition [2] of the invention.

Preferred particulars embodiments of the above mentioned uses and methods include an anti-cancer agent wherein the agent comprises a radionuclide or comprises a drug; or wherein the drug is a drug selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline, such as, for example, bevacizumab, capecitabine, cisplatin, cyclophosphamide, doxorubicin, epirubicin, 5- fluorouracil, folinic acid, methotrexate, or oxaliplatin.

The invention is described below by means of the following examples which are merely illustrative and by no means limiting for the scope of the invention.

EXAMPLES

Materials and Methods

Recombinant human H- and L-apoferritins were purchased from MoliRom, Italy. All chemicals used in this work were purchased from Sigma-Aldrich unless stated otherwise. The liposomal encapsulated doxorubicin (DOX-NP^®) was purchased from Avanti Polar Lipids, Inc. The protein concentration was determined with the Pierce 660 nm Protein Assay (Thermo Scientific). The UV-vis absorption was measured with the NanoDrop 2000c (Thermo Scientific). Biotinylation and streptavidin conjugation

Biotin 3-sulfo-N-hydroxysuccinimide ester sodium salt (NHS-biotin) was used to biotinylate the H- and L-apo ferritin. NHS-biotin in 50-fold molar excess was incubated over night with the 2 mg/ml proteins in lx phosphate buffered saline (PBS) at 4°C. Subsequently, the unbound biotin was removed with the Zeba Spin desalting columns (7 kDa MWCO, Thermo Scientific). The quantification of the biotinylation was carried out with the Pierce Biotin Quantitation Kit (Thermo Scientific). The average biotinylation level was achieved with 14 biotin molecules bound to one L-apoferritin and 20 to one H-apoferritin. For the conjugation, streptavidin was incubated with the purified biotin- apoferritin with the molar ratio 1 : 1 for at least 2 h. Doxorubicin encapsulation

The Dox encapsulation followed the disassembly-reassembly procedure already published (Wong & Mann, 1996, Adv. Mater. 8: 928-32). Briefly, the disassembly of apoferritin was achieved by lowering the pH value of 1 mg/ml apoferritin solution (150 mM NaCl) to 2 with HC1. Disassembled apoferritin was then added in small proportion of 5 μΐ-aliquot into a 20 mg/ml Dox solution with a pH value of 9. The mixture was then incubated for 2 h on ice before the purification of the reassembled apoferritin with the Zeba Spin columns (40 kDa MWCO, Thermo Scientific). The Dox concentration was determined with the absorption at 485 nm with a standard curve.

Cell culture

For the streptavidin delivery, the human colon adenocarcinoma cell line Caco-2 was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and for the Dox delivery the Caco-2 cell line was obtained from the European Collection of Cell Cultures (Sigma-Aldrich, Spain). For the streptavidin delivery the cells (passages 25-38) and for the Dox delivery (passages 20- 48) were cultured in minimum essential medium (MEM; PAA) supplemented with 10% fetal bovine serum (FBS) (Biochrom), 1% nonessential amino acids and 50 μg/ml gentamicin (PAA).

For the assays, Caco-2 cells at 80-90%) confluency were detached with trypsin/EDTA (0.05%>/0.02%o) and seeded onto disposable non-fluorescent multi-well plates at a density of 0.75 x 10⁵ cells/cm² and cultured for 8 days. The culture medium was replaced 1 day after seeding, then every 2 days, and 1 day before the assay. The human liver carcinoma cell line Hep G2 was obtained from the American Type Culture Collection (ATCC, No. HB-8065). The cells between passages 7-16 were used for the experiments. The cells were maintained in MEM supplemented with 10% FBS, 1 mM pyruvate, 1% nonessential amino acids and 50 μg/ml gentamicin. For the assays, Hep G2 cells at 60-70% confluence were detached with trypsin/EDTA (0.05%/0.02%) and seeded onto disposable non- fluorescent multi-well plates at a density of 0.75 x 10⁵ cells/cm² and cultured for 3 days. The culture medium was replaced 1 day after seeding and 1 day before the assay. For the iron challenging, 40 μΜ ferric citrate was added to the medium. The intracellular iron was determined with the Ferene S assay. The cell viability was determined with the cell counting kit-8 (CCK-8) according to the manufacturer's protocol. The absorbance at 450 nm was measured with a plate-reader (Victor X5, PerkinElmer).

Cellular delivery

Before the treatment with ferritin components the differentiated cells were treated with 300 μΜ deferoxamine (DFO) in the medium and the DFO-untreated cells were cultured in normal medium for 24 h. Subsequently, the DFO-containing media were removed and fresh media with ferritin components were added to the cells with 50 μg/ml final ferritin concentration for appropriate time. When not stated otherwise the incubation time was 45 min. In the case of free streptavidin (from Streptomyces avidinii) and Au- streptavidin the streptavidin concentration was 100 μg/ml by the cell treatment.

After the treatment the cells were washed twice with ice cold PBS and continued with the process for the Western blotting or the confocal microscopy.

Multidrug Resistance (MDR)

The multidrug resistance of Caco-2 and Hep G2 cells upon the treatments was measured with Fluorimetric MDR Assay Kits (Abeam). The kit uses a fluorescent MDR indicator for assaying two major MDR pump activities: P-glycoprotein (Pgp, MDRl) and multidrug resistance associated protein (MRPl). The hydrophobic fluorescent dye molecule of the kit rapidly penetrates cell membranes and becomes trapped in cells. By the overexpression of MDRl and/or MRPl this dye will be extruded by the MDR transporters, thus decreasing the cellular fluorescence intensity.

The assay follows the protocol provided by the manufacturer. Briefly, the cells were seeded in a 96-well tissue culture microplate with black wall and clear bottom overnight in 90 μΐ growth medium at 50,000 cells/well. Subsequently, the cells were treated with all composites with a final 5 μg/ml Dox concentration for 18 h. The cells were then washed with PBS and loaded with the MDR dye. After an incubation at room temperature (RT) for 2 h the fluorescence was measured at Ex/Em = 490/525 nm. The blank was measured with the wells treated with the growth medium and the same wash steps. The untreated cells served as the control and the fluorescence intensity of the control was set as 0. The MDR activity was expressed as the decrease of the intensity in percentage compared with the control.

Ferene S assay

For intracellular iron estimation, the cells were washed twice with PBS and a freshly prepared assay solution of 1 mM Ferene S (3-[2-pyridyl]-5,6-bis(2-[-furyl sulfonic acid]-2,4-triazine), 6 M guanidine hydrochloride and 0.5 M ascorbic acid was added to the wells (250 μΕΛνεΙΙ for 24-well plate). The absorbance at 595 nm was measured after incubation for 2 h at room temperature with a plate-reader (Victor X5, PerkinElmer). For the assay with protein samples, the 10 μΐ protein sample was mixed with 150 μΐ assay solution, followed with the same procedure as mentioned above.

Protein detection with Western Blotting The cytoplasmic, membrane and nuclear fractions were collected with the Subcellular Protein Fractionation Kit for Cultured Cells (Thermo Scientific). The total protein concentration of the fractions was measured. 7 μg cytoplasmic, 25 μg nuclear proteins and 20 μg membrane proteins were separated with 4-20% gradient Tris-glycine polyacrylamide gels for SDS-PAGE (Thermo Scientific) and electroblotted onto polyvinylidene fluoride (PVDF) (BioRad). PVDF membranes were blocked for 2 h at room temperature in 5% BSA-Tris-buffered saline/Tween 20 (TBST; 25 mM Tris HC1, pH 7.5/150 mM NaCl/0.05% Tween 20). Next, the membranes were incubated overnight at 4°C with 1 μg/ml rabbit anti-FTHl (151-165) and rabbit anti-FTL for the detection of ferritin. The rabbit anti-streptavidin antibody (Thermo Scientific) was used with the concentration of 2 μg/ml for the detection of streptavidin. The day after, the membranes were washed four times with TBST and incubated at room temperature for 1 h with a peroxidase-conjugated anti-rabbit antibody (Thermo Scientific) in 1 : 1000 as the secondary antibody. For biotin detection, the StrepTactin-HRP was used 1 :50000 (BioRad). The rabbit anti-ferroportin and anti-transferrin receptor antibodies were used in 1 : 1000 for the detection. The rabbit anti-HIF l antibody (Novex^®, Life Technology) was used in 1 :500. The rabbit anti-Na⁺/K⁺ATPase antibody (1 : 15000) and mouse anti- TATA-bingding protein antibody (1 : 1000) from Abeam were used to detect ATPase in membrane fractions and TATA bp in nuclear fractions. The mouse anti-actin antibody (1 : 1000) and the HRP-anti-mouse antibody (1 :50000) (LiCor Bioscience) were used for the actin detection in cytoplasmic fractions. For the enhanced chemiluminescence detection the Pierce ECL Plus Western Blotting Substrate was used and the ECL signal was recorded with the C-DiGit Blot Scanner (Li-Cor Bioscience).

Laser-scanning Confocal Microscopy

For study of distribution and translocation of endogenous and exogenous ferritin, Caco- 2 cells were grown on a 4- or 8-well chamber slide with a cell density of 50000 cells/cm² and the assays were carried out after 8 days growth.

Live cell imaging:

Cells were cultured in 35 mm petri dish and grown to approximately 60 % confluency. The cell culture medium was changed with live cell imaging solution (Gibco). 10 μg/ml H-Dox, 20 μg/ml free or liposomal Dox was added to the solution. Living images of cell culture were taken with an argon ion UV laser for excitation and emissions of Dox (Ex/Em: 488/595-650 nm, red). During the imaging period (ca. 2 h) the alignment was kept constant.

Ferritin-conjugated streptavidin:

Cells were washed with ice cold PBS, then fixed and permeabilized with 4% paraformaldehyde containing 0.05% Triton-X-100 for 30 min. Excess of paraformaldehyde was removed by incubation with 0.1 mM NH₄CI for 5 min. After blocking with 5% bovine serum albumin in PBS, cells were incubated for 2 h with the primary antibody rabbit anti- Streptavidin antibody diluted 1 :200 in 5% bovine serum albumin in PBS. The second antibody anti-rabbit IgG-FITC diluted 1 :200 in PBS was incubated for 1 h. Cell nuclei were stained with 5 μg/ml PI (propidium iodide in PBS) for 5 min. Coverslips were mounted with the CitiFlour Solid Mounting Kit (Agar Scientific). Samples were examined with a laser scanning confocal microscopy (Leica TCS SP2 DM IRE2) and imaged with a HCX PL APO CS 40 x 1.25 oil immersion objective (Leica Microsystems, Wetzlar, Germany). The 488 nm-line from an argon ion UV laser was used for excitation of FITC and PI, and emissions were collected at 515 nm-530 nm (FITC, green), and 630 nm-650 nm (PI, red). Channel sequential scanning mode was used. Overlay pictures of PI channel (red) and FITC channel (green) are shown. Scale bars: 20 μιη

Apoferritin-encapsulated Dox:

Cells were washed with ice cold PBS, then fixed and permeabilized with 4% paraformaldehyde containing 0.05% Triton-X-100 for 30 min. Excess of paraformaldehyde was removed by incubation with 0.1 mM NH₄CI for 5 min. Cell nuclei were stained with 10 μg/ml DAPI (4',6-diamidino-2-phenylindole dihydrochloride in PBS) for 10 min. Coverslips were mounted with the CitiFlour Solid Mounting Kit (Agar Scientific).

Samples were examined with the laser scanning confocal microscopy (Zeiss LSM 710) and imaged with a 63x objective. The lines from an argon ion UV laser were used separately for excitation of DAPI (405 nm), FITC (488 nm) and Dox (488 nm), and emissions were collected at 435 nm-450 nm (DAPI, blue), 515 nm-530 nm (FITC, green), and 595 nm-650 nm (Dox, red). Channel sequential scanning mode was used. Overlay pictures of DAPI channel (blue) and Dox channel (red), or DAPI channel (blue) and FITC channel (green) are shown.

Statistic analysis

All values of in vitro tests were expressed as the mean ± SD. The significance was analyzed with one-way ANOVA. The comparison between groups was performed with the unpaired two-tailed Student's t-test.

EXAMPLE 1

Intracellular distribution of ferritin H-chains and L-chains

Under normal condition the H-chain ferritin was distributed both in cytosol and in nuclei (Fig. la), which confirms a natural presence of nuclear ferritin in Caco-2 cells. The iron chelator deferoxamine (DFO), which was reported to be able to reduce the nuclear ferritin, decreased also significantly the amount of the total and, especially, the nuclear ferritin in Caco-2 cells (Fig. la). On the Western blot the H-chains were detectable with the loading amount of the cytoplasmic fractions only when human H- type apoferritin (biotin-H) was supplemented to the cells, even without the DFO- pretreatment (Fig. lb and Fig. 2). In the contrast, H-chain was detected unequally in nuclear fractions by all three treatments with the highest amount by the H-strep supplemented cells. The enhanced nuclear translocation of H-ferritin upon the DFO pretreatment indicates that the ferritin delivery is influenced by cellular iron status. The pervasive presence of H-chains of ferritin in the nuclear fractions indicated that either the nuclear ferritin was not totally eliminated by DFO treatment or the cells recovered the nuclear ferritin rapidly as soon as DFO was removed from the medium. Biotin was detected to identify the exogenous biotinylated ferritin in fractions. The cells apparently uptake both exogenous H- and L-ferritin, which have a molecular weight difference of approximate 2 kDa (Fig. lb and Fig. 2). It was also found that the cellular uptake and the nuclear translocation of exogenous apoferritin was a rapid process (Fig. lc). Within 15 minutes a significant amount of the supplemented biotin-H was internalized and translocated into the nuclei. After 30 minutes the nuclear presence of the exogenous ferritin decreased while its concentration in cytoplasm remained constant for at least 24 hours (Fig. lc). Being analogous to the endogenous protein, the exogenous ferritin obeys some natural cytological regulations, which may avoid the bio-incompatibility due to an uncontrolled cellular accumulation. For the intended nuclear delivery of loaded drugs, a nuclear translocation within the intact 24-meric cage structure of ferritin is particularly desired to avoid unnecessary cytoplasmic leakage and degradation or the drug resistance related efflux. However, it is unclear in which form the H-chain is translocated into nuclei.

Upon the supplement of biotinylated L-chain (biotin-L) or the simultaneous supplement of biotin-L (all ferritin L-chains) and -H (all ferritin H-chains) in equal ratio, no biotin-L was detected in the nuclei (Fig. lb and Id). Considering the absence of the L-chain in the nuclei and the fact that 24-meric ferritin can be formed from L- and H-chain proteins in any proportion, cells were treated with ferritin assembled from both types of biotinylated ferritin subunits in 1 : 1 ratio (biotin-L/H). Indeed, the nuclear-translocation of L-chains only occurred through the L/H-chimeric apoferritin (Fig. Id). This observation strongly supports the nuclear translocation of ferritin in its intact spherical structure, since a cytoplasmic disassembly of the L/H-ferritin cage will lead to an H- chain translocation only. The intact ferritin complex is smaller than the upper limit for an active transport into nuclei, thus drugs encapsulated inside H-chain-containing apoferritin can be delivered into nuclei without unnecessary cytoplasmic release (Fig. le).

EXAMPLE 2

Nuclear delivery of doxorubicin encapsulated in H-ferritin

The rapid nuclear translocation through an intact ferritin cage is unique for the delivery of drugs executing functions inside nuclei, such as the anti-cancer drug, doxorubicin (Dox). Human intestinal Caco-2 and liver Hep G2 cells were treated with Dox encapsulated in the cavity of H- ferritin (H-Dox) (Fig. 3). Already within 15 minutes treatment with H-Dox a rapid delivery of Dox into the nuclei without cytoplasmic accumulation was achieved (Fig. 4a and 4b).

This is in contrast to the treatment with free Dox or with Dox encapsulated in pegylated liposomes (Lipo-Dox) (similar to the clinical drug Doxil^®) (Fig. 4a and 4b). Once delivered, nuclear Dox successfully intercalated into DNA (Fig. 5). A nuclear accumulation of Dox was barely detectable with Lipo-Dox, even upon 5-fold concentration increase. Dox cannot be detected in the cell nuclei within the same time when the cells were treated with the uncapsulated drug of the same concentration. At least two hours with double concentration of free Dox were necessary to reach a similar accumulation in the nuclei (Fig. 6). The ferritin shuttled delivery clearly facilitates the nuclear transport that directly lowers the drug dose and reduces the exposure time.

EXAMPLE 3

Nuclear delivery of streptavidin conjugated to H- ferritin

For an organ- or tissue-specific targeted delivery in vivo, further modifications of the outer surface of ferritin may be necessary. The nuclear translocation of apoferritin does not need a nuclear localization signal (NLS), but it is unclear whether a modified outer surface would be a hindrance. It was demonstrated that the biotinylation did not retard the cellular uptake and the nuclear translocation of apoferritin. In order to evaluate the impact of additional modifications on the apoferritin-assisted nucleocytoplasmic delivery, streptavidin of 60 kDa MW was bound through biotin to apoferritin. Again, the nuclear translocation of streptavidin was only achievable with H-chain conjugation, while the free and L-chain conjugated streptavidin remained in the cytoplasm (Fig. 4c and Fig. 7). The different delivery destination for the conjugated streptavidin with the H- and L-chains is consistent with the described cellular distribution of endogenous ferritin in the distinctive cellular compartments. Confocal fluorescence microscopy further confirmed the distribution of streptavidin being consistent with the described distribution of endogenous ferritin in distinct cellular compartments (Fig. 4d and Fig. 8- 10).

Besides ferritin, gold nanoparticles of 10 nm in size were also tested for the delivery. Since the cellular uptake of nanoparticles is unlikely dependent on the cellular iron level, the cellular uptake streptavidin was not influenced by the DFO. Free streptavidin or streptavidin conjugated to gold nanoparticles (10 nm), although being smaller than ferritin (12 nm), cannot be translocated into the nuclei (Fig. 4d), confirming that the nuclear translocation of streptavidin follows the cyto logical transport of apoferritin and designates an advantage of ferritin as the delivery agent. The unchanged nucleocytoplasmic delivery pattern, even with surface modifications (different in size, such as biotin and streptavidin), makes apoferritin ultimately promising as drug delivery agent for clinical applications. EXAMPLE 4

H-ferritin-encapsulated doxorubicin bypasses the cellular multidrug resistance

Cytoplasmic exposure of drugs can activate several resistance mechanisms, such as, drug efflux and enzymatic inactivation. Once encapsulated, the cells upon uptake recognize only the ferritin but not the Dox inside. Expectedly, free Dox activated the multidrug resistance (MDR) mechanism of both Caco-2 and Hep G2 cells (Fig. 11a). However, the MDR activation was not observed upon treatment with Dox encapsulated in either liposomes or apoferritin. Bypassing the MDR activation, the apoferritin- mediated nuclear delivery improves the antineoplastic effect of the drug.

EXAMPLE 5

Iron regulation mediated by H-ferritin-encapsulated doxorubicin

The clinical use of Dox is seriously restricted for its cardio toxicity, caused by its cellular iron dysregulation effects. Indeed, the cellular iron uptake was dysregulated upon treatment with free Dox (Fig. 1 lb). The hampered iron uptake by Caco-2 cells and elevated uptake by Hep G2 cells were mainly due to the Dox-induced up- or down- regulated expression of the transferrin-receptor (TfR), respectively (Fig. 11c). Since the intestinal cells are responsible for the iron uptake and the liver cells for its storage, Dox- induced organ-specific iron dysregulation may be the reason for simple iron depletion failing to achieve cardioprotection in some cases. The altered iron uptake induced by Dox was attenuated when the cells were treated with H-Dox (Fig. l ib). Protective effects have been observed with Dox-induced expression of endogenous ferritin in cells and animal models. The results shown here indicate that the intrinsic cellular iron regulation functions of exogenous and endogenous apoferritin are comparable. Besides restoring the changes of TfR caused by Dox, apoferritin also upregulated the only identified cellular iron exporter in Hep G2 cells, ferroportin (Fig. 11c).

EXAMPLE 6

Suppression of HIF-la upregulation in tumor cells Free Dox further induces the accumulation of HIF-Ια in Hep G2, but not Caco-2 cells (Fig. l id). The upregulated HIF-Ια in tumor cells stimulates the resistance to anticancer drugs and causes poor chemotherapeutic response. HIF-Ια inhibition or down- regulation is suggested for enhanced chemotherapeutic efficiency. H-Dox avoided the HIF-Ια up-regulation in Hep G2 cells and did not induce additional HIF-Ια down- regulation in Caco-2 cells (Fig. l id). These results indicate that the apoferritin-Dox combination has a synergistic anti-cancer effect besides the efficient nuclear delivery.

EXAMPLE 7

H-ferritin exhibits low toxicity

With its autogenic origin the delivery agent ferritin is expected to show low cytotoxicity (Fig. 12) and cause less immune response.

The capability of apoferritin to deliver both surface-conjugated (assuming biotin or streptavidin being kinds of drugs or agents) and cavity-loaded drugs and the intrinsic nucleocytoplasmic selectivity of L- and H-chains signify its versatility for targeted delivery. The cellular regulation of the nuclear translocation of endogenous ferritin by various cytokines indicates that the ferritin-based delivery may be a pathway, fine- tunable and controllable from the very beginning of its cellular contact, the receptor- mediated uptake. Different to the currently utilized NLS-directed nuclear transport (Pollard, V. W. et al. A novel receptor-mediated nuclear protein import pathway. Cell 86, 985-994 (1996)), an active NLS-independent nuclear translocation of exogenous apoferritin provides a novel mechanism for drug delivery.

Claims

1. Use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for delivering the agent to the nucleus of a target cell.

2. A method for delivering an agent to the nucleus of a target cell which comprises contacting a ferritin nanoparticle comprising at least one ferritin H-chain and an agent with a target cell under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell.

3. Use or method according to any of claims 1 or 2, wherein the delivering of the agent to the nucleus of a target cell is performed in vitro or in vivo.

4. Use or method according to any of claims 1 or 3, wherein the ferritin is human ferritin.

5. Use or method according to any of claims 1 or 4, wherein the ferritin is apoferritin.

6. Use or method according to any of claims 1 or 5, wherein the apoferritin is human apoferritin.

7. Use or method according to any of claims 1 to 6, wherein the ferritin nanoparticle consists of 24 ferritin H-chains.

8. Use or method according to any of claims 1 to 7, wherein the ferritin H-chain is human ferritin H-chain.

9. Use or method according to any of claims 1 to 8, wherein the agent comprises a protein, a peptide, a nucleic acid, a small molecule, or a metal.

10. Use or method according to any of claims 1 to 9, wherein the agent comprises a hormone, an imaging agent, or a drug.

11. Use or method according to any of claims 1 to 10, wherein the ferritin nanoparticle further comprises a second agent.

12. Use or method according to claim 11, wherein the second agent comprises a protein, a peptide, a nucleic acid, a small molecule, or a metal.

13. Use or method according to any of claims 11 or 12, wherein the second agent comprises a hormone, an imaging agent, or a drug.

14. Use or method according to any of claims 1 to 14, wherein the agent comprises an imaging agent.

15. Use or method according to any of claims 1 to 14, wherein the agent comprises a drug.

16. Use or method according to any of claims 1 to 15, wherein the agent is a drug selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline.

17. Use or method according to any of claims 1 to 16, wherein the agent is a drug and said drug is doxorubicin.

18. Use or method according to any of claims 1 to 17, wherein the agent is a drug and said drug is bevacizumab, capecitabine, cisplatin, cyclophosphamide, epirubicin, 5- fluorouracil, folinic acid, methotrexate, or oxaliplatin.

19. Use or method according to any of claims 1 to 18, wherein the target cell is a tumour cell.

20. Use or method according to any of claims 1 to 19, wherein the target cell is a tumour cell selected from the group consisting of a carcinoma cell, a sarcoma cell, a lymphoma cell, a leukemic cell, a cell from a germ cell tumour, and a blastoma cell.

21. A ferritin nanoparticle comprising at least one ferritin H-chain and an agent for use in the treatment of a disease, wherein said agent is an agent that is indicated for the treatment of said disease.

22. Use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent for the manufacture of a pharmaceutical composition the treatment of a disease, wherein said agent is an agent that is indicated for the treatment of said disease.

23. A method for the treatment of a disease which comprises administering to a subject in need thereof a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent is an agent that is indicated for the treatment of said disease.

24. Ferritin nanoparticle for use according to claim 21, use according to claim 22 or method according to claim 23, wherein the ferritin is human ferritin.

25. Ferritin nanoparticle for use according to claim 21 or 24, use according to claim 22 or 24, or method according to claim 23 or 24, wherein the ferritin is apoferritin.

26. Ferritin nanoparticle for use according to any of claims 21, 24 or 25, use according to any of claims 22, 24 or 25, or method according to any of claims 23, 24 or 25, wherein the ferritin is human apoferritin.

27. Ferritin nanoparticle for use according to any of claims 21, or 24-26, use according to any of claims 22, or 24-26, or method according to any of claims 23-26, wherein the ferritin nanoparticle consists of 24 ferritin H-chains.

28. Ferritin nanoparticle for use according to any of claims 21, or 24-27, use according to any of claims 22, or 24-27, or method according to any of claims 23-27, wherein the ferritin H-chain is human ferritin H-chain.

29. Ferritin nanoparticle for use according to any of claims 21, or 24-28, use according to any of claims 22, or 24-28, or method according to any of claims 23-28, wherein the ferritin nanoparticle further comprises a second agent.

30. Ferritin nanoparticle for use according to any of claims 21, or 24-29, use according to any of claims 22, or 24-29, or method according to any of claims 23-29, wherein the agent comprises a radionuclide or a drug.

31. Ferritin nanoparticle for use according to any of claims 21, or 24-30, use according to any of claims 22, or 24-30, or method according to any of claims 23-30, wherein the agent is a drug selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline.

32. Ferritin nanoparticle for use according to any of claims 21, or 24-31, use according to any of claims 22, or 24-31 , or method according to any of claims 23-31, wherein the agent is a drug and said drug is bevacizumab, capecitabine, cisplatin, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, folinic acid, methotrexate, or oxaliplatin.

33. Ferritin nanoparticle for use according to any of claims 21, or 24-32, use according to any of claims 22, or 24-32, or method according to any of claims 23-32, wherein the disease is a cancer.

34. A ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for use in in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

35. Use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for in vivo delivering the agent to the nucleus of a target cell, or for in vivo visualizing the nucleus of a target cell.

36. Use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, in the manufacture of a pharmaceutical composition for in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

37. An in vivo method for delivering an agent to the nucleus of a target cell in a subject, or for in vivo visualizing the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle comprising at least one ferritin H-chain and an agent with a target cell, wherein the agent comprises an imaging agent.

38. Ferritin nanoparticle for use according to claim 34, use according to claim 35 or 36, or method according to claim 37, wherein the ferritin is human ferritin.

39. Ferritin nanoparticle for use according to claim 34 or 38, use according to claim 35, 36 or 38, or method according to claim 37 or 38, wherein the ferritin is apoferritin.

40. Ferritin nanoparticle for use according to any of claims 34, 38 or 39, use according to any of claims 35, 36, 38 or 39, or method according to any of claims 37, 38 or 39, wherein the ferritin is human apoferritin.

41. Ferritin nanoparticle for use according to any of claims 34, or 38-40, use according to any of claims 35, 36, or 38-40, or method according to any of claims 37-40, wherein the ferritin nanoparticle consists of 24 ferritin H-chains.

42. Ferritin nanoparticle for use according to any of claims 34, or 38-41, use according to any of claims 35, 36, or 38-41, or method according to any of claims 37-41, wherein the ferritin H-chain is human ferritin H-chain.

43. Ferritin nanoparticle for use according to any of claims 34, or 38-42, use according to any of claims 35, 36, or 38-42, or method according to any of claims 37-42, wherein the ferritin nanoparticle further comprises a second agent.

44. Ferritin nanoparticle for use according to any of claims 34, or 38-43, use according to any of claims 35, 36, or 38-43, or method according to any of claims 37-43, wherein the agent is a radionuclide, a fluorophore or a magnetic contrast agent.

45. An in vitro use of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein the agent comprises an imaging agent, for delivering the agent to the nucleus of a target cell, or for visualising the nucleus of a target cell.

46. An in vitro method for delivering an agent to the nucleus of a target cell, or for visualising the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle comprising at least one ferritin H-chain and said agent with a target cell, wherein the agent comprises an imaging agent, under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell.

47. A ferritin nanoparticle comprising at least one ferritin H-chain and an agent with the proviso that:

the agent is not a metal,

if the agent is doxorubicin, then the ferritin nanoparticle does not consist of 24 ferritin H-chains,

48. Ferritin nanoparticle according to claim 47, wherein the ferritin is human ferritin.

49. Ferritin nanoparticle according to claim 47 or 48, wherein the ferritin is apoferritin.

50. Ferritin nanoparticle according to claim 49, wherein the apoferritin is human apoferritin.

51. Ferritin nanoparticle according to any of claims 47 to 50, wherein the ferritin nanoparticle consists of 24 ferritin H-chains.

52. Ferritin nanoparticle according to any of claims 47 to 51, wherein the ferritin H- chain is human ferritin H-chain.

53. Ferritin nanoparticle according to any of claims 47 to 52, wherein the agent comprises a protein, a peptide, a nucleic acid, or a small molecule.

54. Ferritin nanoparticle according to any of claims 47 to 53, wherein the agent comprises a hormone, an imaging agent, or a drug.

55. Ferritin nanoparticle according to any of claims 47 to 54, wherein the ferritin nanoparticle further comprises a second agent.

56. Ferritin nanoparticle according to claim 55, wherein the second agent comprises a protein, a peptide, a nucleic acid, or a small molecule.

57. Ferritin nanoparticle according to claim 55 or 56, wherein the second agent comprises a hormone, a non-metal imaging agent, or a drug.

58. Ferritin nanoparticle according to any of claims 47 to 57, wherein the agent comprises a non-metal imaging agent or a drug.

59. Ferritin nanoparticle according to any of claims 47 to 58, wherein the agent is a drug selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline.

60. Ferritin nanoparticle according to any of claims 47 to 59, wherein the agent is a drug and said drug is bevacizumab, capecitabine, cisplatin, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, folinic acid, methotrexate, or oxaliplatin.

61. A ferritin nanoparticle according to any of claims 47 to 60 for use in the treatment of a disease, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

62. Use of a ferritin nanoparticle according to any of claims 47 to 60 for the manufacture of a pharmaceutical composition for the treatment of a disease, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

63. A method for the treatment of a disease which comprises administering to a subject in need thereof a ferritin nanoparticle according to any of claims 47 to 60, wherein said ferritin nanoparticle comprises an agent and said agent is an agent that is indicated for the treatment of said disease.

64. Ferritin nanoparticle for use according to claim 61 , use according to claim 62, or method according to claim 63, wherein the agent comprises a radionuclide or a drug.

65. Ferritin nanoparticle for use according to any of claims 61 or 64, use according to any of claims 62 or 64, or method according to any of claims 63 or 64, wherein the agent is a drug selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline.

66. Ferritin nanoparticle for use according to any of claims 61, 63 or 64, use according to any of claims 62, 64 or 65, or method according to any of claims 63, 64 or 65, wherein the disease is a cancer.

67. A ferritin nanoparticle according to any of claims 47 to 60, wherein the agent comprises a non-metal imaging agent, for use in in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

68. Use of a ferritin nanoparticle according to any of claims 47 to 60, wherein the agent comprises a non-metal imaging agent, for in vivo delivering the agent to the nucleus of a target cell, or for in vivo visualizing the nucleus of a target cell.

69. Use of a ferritin nanoparticle according to any of claims 47 to 60, wherein the agent comprises a non-metal imaging agent, in the manufacture of a pharmaceutical composition for in vivo delivering the agent to the nucleus of a target cell, or for use in in vivo visualizing the nucleus of a target cell.

70. An in vivo method for delivering an agent to the nucleus of a target cell in a subject, or for in vivo visualizing the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle according to any of claims 47 to 60, wherein the agent comprises a non-metal imaging agent.

71. Ferritin nanoparticle for use according to claim 67, use according to any of claims 68 or 69, or method according to claim 70, wherein the ferritin nanoparticle further comprises a second agent.

72. Ferritin nanoparticle for use according to any of claims 67 or 71, use according to any of claims 68, 69 or 71 , or method according to any of claims 70 or 71, wherein the target cell is a tumoural cell.

73. An in vitro use of a ferritin nanoparticle according to any of claims 47 to 60, wherein the agent comprises a non-metal imaging agent for delivering the agent to the nucleus of a target cell, or for visualising the nucleus of a target cell.

74. An in vitro method for delivering an agent to the nucleus of a target cell, or for visualising the nucleus of a target cell, the method comprising contacting a ferritin nanoparticle according to any of claims 47 to 60, wherein the agent comprises a non-metal imaging agent, with a target cell, wherein the agent comprises a non- metal imaging agent, under conditions that allow the internalisation of the ferritin nanoparticle by the target cell and further translocation of the ferritin nanoparticle into the nucleus of the target cell.

75. A pharmaceutical composition, selected from: c) a pharmaceutical composition comprising a therapeutically effective amount of a ferritin nanoparticle comprising at least one ferritin H-chain and an agent, wherein said agent is an agent indicated for the treatment of a disease, together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle; and d) a pharmaceutical composition comprising a therapeutically effective amount of a ferritin nanoparticle according to any of claims 47 to 60, wherein the agent is an agent indicated for the treatment of a disease, together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle.

76. A composition comprising, separately, (i) a ferritin nanoparticle comprising at least one ferritin H-chain and (ii) an anti-cancer agent.

77. A pharmaceutical composition comprising, separately, (i) a pharmaceutical composition comprising a ferritin nanoparticle comprising at least one ferritin H- chain together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle, and (ii) a pharmaceutical composition comprising an anti-cancer agent together with a pharmaceutically acceptable excipient, carrier, adjuvant, or vehicle.

78. Composition according to claim 76 or 77, wherein the ferritin is human ferritin.

79. Composition according to any of claims 76 to 77, wherein the ferritin is apoferritin.

80. Composition according to any of claims 76 to 79, wherein the apoferritin is human apoferritin.

81. Composition according to any of claims 76 to 80, wherein the ferritin nanoparticle consists of 24 ferritin H-chains.

82. Composition according to any of claims 76 to 81, wherein the ferritin H-chain is human ferritin H-chain.

83. Composition according to any of claims 76 to 82, wherein the anti-cancer agent is a radionuclide or a drug.

84. Composition according to any of claims 76 to 83, wherein the anti-cancer agent is a drug selected from the group consisting of an alkylating agent, an antimetabolite, a topoisomerase inhibitor and an anthracycline.

85. Composition according to any of claims 76 to 84, wherein the anti-cancer agent is bevacizumab, capecitabine, cisplatin, cyclophosphamide, doxorubicin, epirubicin, 5-fluorouracil, folinic acid, methotrexate, or oxalip latin.

86. A composition according to any of claims 76 to 85 for use in the treatment of cancer.

87. Use of the composition according to any of claims 76 to 85 in the manufacture of a medicament comprising separately a (i) a pharmaceutical composition comprising a ferritin nanoparticle comprising at least one ferritin H chain and (ii) a pharmaceutically composition comprising an anti-cancer agent.

88. A method for the treatment of cancer which comprises administering to a subject in need thereof of a composition according to any of claims 76 to 85.