WO2011070212A2 - Multifunctional nanostructures as trimodal mri/oi/spect diagnosis agents - Google Patents

Multifunctional nanostructures as trimodal mri/oi/spect diagnosis agents Download PDF

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WO2011070212A2
WO2011070212A2 PCT/ES2010/070816 ES2010070816W WO2011070212A2 WO 2011070212 A2 WO2011070212 A2 WO 2011070212A2 ES 2010070816 W ES2010070816 W ES 2010070816W WO 2011070212 A2 WO2011070212 A2 WO 2011070212A2
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ferritin
solution
peg
quantum dot
magnetoferritin
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PCT/ES2010/070816
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Spanish (es)
French (fr)
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WO2011070212A3 (en
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José Manuel DOMÍNGUEZ VERA
Natividad GÁLVEZ RODRÍGUEZ
Belén FERNÁNDEZ LÓPEZ
Elsa Valero Romero
Federico Boschi
Laura Calderan
Pasquina Marzola
José Juan CALVINO GÁMEZ
Ana B. HUNGRÍA HERNÁNDEZ
Rafael Cuesta Martos
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Universidad De Granada
Universidad De Cádiz
Universidad De Jaén
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0002—General or multifunctional contrast agents, e.g. chelated agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0065—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle
    • A61K49/0067—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the luminescent/fluorescent agent having itself a special physical form, e.g. gold nanoparticle quantum dots, fluorescent nanocrystals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A61K49/1821—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles
    • A61K49/1824—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles
    • A61K49/1827—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle
    • A61K49/1866—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles coated or functionalised microparticles or nanoparticles coated or functionalised nanoparticles having a (super)(para)magnetic core, being a solid MRI-active material, e.g. magnetite, or composed of a plurality of MRI-active, organic agents, e.g. Gd-chelates, or nuclei, e.g. Eu3+, encapsulated or entrapped in the core of the coated or functionalised nanoparticle the nanoparticle having a (super)(para)magnetic core coated or functionalised with a peptide, e.g. protein, polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to a multifunctional nanostructure, specifically a ferritin, in addition to its application as a contrast agent in OI, MRI or SPECT. Therefore, the invention could be framed within the field of biomedicine.
  • Nanotechnology in biotechnology has made a new discipline flourish: nanomedicine.
  • metal nanoparticles are designed and prepared to obtain bio-images through the simultaneous use of several techniques, effective distribution of drugs or techniques of promising therapies such as hyperthermia caused locally by magnetic nanoparticles. It is an area of tremendous potential subject to the development of new nanostructures for its advancement.
  • Magnetic nanoparticles have attracted attention primarily because of their potential use as contrast agents in Magnetic Resonance Imaging (MRI). This technique is based on the magnetic resonance of the protons of body tissues (water, membranes, lipids, proteins, etc.) and is currently the most powerful method of diagnosis.
  • MRI Magnetic Resonance Imaging
  • the contrast in MRI can be improved with paramagnetic substances.
  • the ability of a compound to increase the relaxation rate of proton spins of surrounding water molecules is called relaxation and is defined as R1 -1 / T1 or R2-1 / T2.
  • Superparamagnetic nanoparticles are candidates to act as contrast agents in MRI. Like paramagnetic substances, they lose their magnetization when the external magnetic field is removed, but unlike them, their magnetization is significantly higher. Therefore, the relaxation they produce is much more high than those of the classic paramagnetic complexes of Gd (lll).
  • the QDs are inorganic nanoparticles, generally composed of elements of groups ll-VI and lll-V, which, due to their quantum confinement of charges in a tiny space show unique fluorescent properties: narrow emission spectra, high quantum yield, Wide absorption spectra, good chemical stability and high photostability and emission wavelength dependent on size, extending its emission range to the NIR (near infrared) or IR (infrared) region and offering greater tissue penetration for a better image .
  • these nanoparticles need to be functionalized with some type of polymer or protein that makes them biocompatible and therefore suitable for use in vivo.
  • MRI-OI magnetic resonance imaging
  • the magnetic nanocomponent can incorporate a radiolabel, such as 99m Tc0 4 " , adding a new medical imaging modality to the multifunctional nanostructure by detecting the gamma radiation that the radionuclide generates by scintigraphy (SPECT).
  • SPECT scintigraphy
  • a key point for the biomedical application of metal nanoparticles is that they cannot be toxic and must remain in circulation long enough to reach the biological target.
  • the mononuclear phagocytic system (MPS) recognizes and eliminates, through macrophages, the particles of the circulation, with their simultaneous concentration in organs with high phagocytic activity (mainly the liver). Therefore, a sensible strategy is the use of nanoparticles that are able to evade the attack by the MPS and are not phagocytosed by macrophages, consequently increasing the half-life in plasma and allowing to reach a specific organ or tissue.
  • This type of diagnostic agents ideal for clinical application should have a high and specific accumulation in the appropriate cells, which would result in the possibility of diagnosing an even treatable disease more and more early.
  • a possible route to obtain metallic nanoparticles without aggregation and with controlled size is the use of a pre-organized molecular platform, with a cavity that can act as a nanoreactor for chemical and spatial control in the formation of nanoparticles.
  • a typical example of this type of molecules is the ferritin protein.
  • Apoferritin consists of a spherical protein formed by 24 subunits surrounding an aqueous cavity with a diameter of approximately 8 nm.
  • an objective of the present invention is to provide an OI contracting agent, which can also be used as a bimodal agent, OI-MRI, and even trimodal, OI-MRI-SPECT, based on a ferritin, characterized in that it comprises at least one quantum dot (QD) and at least one molecule of a biocompatible polymer both covalently bonded to the surface of the ferritin (Figure 1).
  • QD quantum dot
  • Figure 1 a biocompatible polymer
  • the ferritin of the invention is biocompatible and surprisingly bioconjugate with a quantum dot, significantly improves the biodistribution of both particles separately.
  • the nanostructure formed by the conjugation of ferritin and quantum dot can be used as a contrast agent in RO and shows sufficiently long lifespan to be distributed through the circulatory system without being phagocyted in less than 3 hours, but at the same time Short enough to prevent its accumulation in the body.
  • ferritin is understood as any apophritin, independent of the material that is encapsulated in its internal cavity, including the apo-ferritins which the internal cavity is empty.
  • the fluorescent components such as QD525, QD655 and QD800, which could covalently bind to the outer layer of the ferritin by forming a covalent amide bond by reaction between the -NH 2 groups free of the lysine residues of the outer layer of ferritin and the carboxylic groups of QD.
  • quantum dots QDs have been used successfully as new fluorescent markers in the biomedical field and are considered as a promising tool in Optical Imaging for in vivo clinical diagnosis [Choi, H. _S. Liu, W. Misra, W., Tanaka, E., Zimmer, JP, Ipe, BI, Bawendi, MG, Frangioni, JV, Nal Biotechnol. 2007, 25, 1165-1170 and Qi, L, Gao, X. Expert. Opin. Drug Delivery 2008, 5, 263-267.].
  • the QDs are inorganic nanoparticles, generally composed of elements of groups ll-VI and lll-V, which, due to their quantum confinement of charges in a tiny space show unique fluorescent properties: narrow emission spectra, high quantum yield, Wide absorption spectra, good chemical stability and high photostability and emission wavelength dependent on size, expanding its emission range and offering greater tissue penetration for a better image.
  • One of the limitations of the QD to be able to have applications in vivo is their low residence time in blood. However, this limitation can be overcome by functionalization with hydrophobic polymers such as polyethylene glycol (PEG) [Daou, T. Jean; Li, Liang; Reiss, Peter; Josserand, Veronique; Texier, Isabelle.
  • PEG polyethylene glycol
  • the quantum dots can be selected, for example, from those listed in the Invitrogen Molecular Probes 2009 catalog and preferably are selected from Qdot® 625 ITK TM carboxyl quantum dots (A1 0200), Qdot® 605 ITK TM carboxyl quantum dots 8 ⁇ solution ( Q21301 MP), Qdot® 585 ITK TM carboxyl quantum dots 8 ⁇ solution (Q21 31 1 MP), Qdot® 655 ITK TM carboxyl quantum dots 8 ⁇ solution (Q21 321 MP), Qdot® 565 ITK TM carboxyl quantum dots 8 ⁇ solution (Q21 331 MP), Qdot® 525 ITK TM carboxyl quantum dots 8 ⁇ solution (Q21 341 MP), Qdot® 705 ITK TM carboxyl quantum dots 8 ⁇ solution (Q21 361
  • Preferred quantum dots are those that emit between 750 and 850
  • R FE is the radius of the nanoparticle
  • R QD It is the radius of the quantum dot (QD).
  • This expression estimated, it comes from dividing the area of the ferritin covered by the quantum dots and dividing it by the area of ferritin covered by a single quantum dot.
  • the calculation assumes a compact packing of QDs on the surface of the ferritin and takes into account the gaps between the QDs.
  • the maximum number of QDs increases from 45 to 133 when the diameter of the QD decreases from 4 to 2 nm (D. Wang, J. He, N. Rosenzweig, Z. Rosenzweig, Nano Lett. 2004, 4, 409-413).
  • ferritin has chains of a biocompatible polymer covalently bonded to the surface of ferritin.
  • the presence of this polymer improves the properties of the ferritin of the invention to be used as a contrast agent since it increases its overall stability, obtaining adequate average life times in the blood for use.
  • the preferred biocompatible polymer is polyethylene glycol (PEG), among other reasons for its industrial availability, its ease of incorporation to the surface of ferritin and its high biocompatibility.
  • PEG polyethylene glycol
  • the process of covalently binding the PEG polymer to other molecules, usually drugs or therapeutic proteins, is known as PEGylation (Kohler, N.; Fryxell, GE; Zhang, MJ Am. Chem. Soc. 2004, 126, 7206.
  • PEG is covalently bound to ferritin.
  • This process can be carried out easily, by incubating a derivative of the reactive PEG with the nanoparticles.
  • the covalent union of PEG masks them of the immune system, increases hydrodynamic size (size in solution) which increases the half-life in blood and reduces their elimination by the immune system.
  • This process also increases the water solubility of said nanoparticles and generally gives it additional stability.
  • the number of PEG molecules that bind to the surface of the nanoparticle can also be controlled.
  • PEG derivatives of the succinimidyl ester type allow the formation of a covalent bond of the amide type by reaction with the amino groups on the outer surface of the nanoparticles.
  • PEG must be derivatized with functional groups capable of binding on their own or with the aid of a reagent to the surface of ferritin.
  • the PEG is preferably linked by amide bonds, therefore the preferred PEG derivatives, as mentioned above, contain the succinimidyl ester functional group, which allows a direct functionalization of the ferritin, since this activated ester group It reacts with the amino groups surrounding the nanoparticles, forming the covalent bond type amide.
  • PEG1 1 63 MeO-PEG-COO-Su a-Methoxy-cjo-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton.
  • PEGs functionalized as succinimidyl ester are commercial and their covalent coupling to the superparamagnetic particle can be carried out after the introduction of 99m Tc.
  • the number of PEG chains introduced can be controlled depending on the stoichiometry and PEG used. For example, when activated ester derivatives are used, given the high reactivity of said esters and the amines of the nanoparticle, the reaction is complete, such that the number of PEG covalently bonded to the surface of the nanoparticle coincides with the number of molecules of the PEG derivative / nanoparticles that is used in the reaction.
  • the number of chains introduced was checked through the use of an electrophoretic pattern, analyzing the different ferritins derivatized with PEG and seeing their correspondence with a gradual and gradual increase in molecular weight and therefore a greater retention.
  • the PEG can be mono- or bifunctionalized with activated esters and PEG can be joined with different density and crystallinity.
  • the ferritin stability of the invention is enhanced by the introduction of the biocompatible polymer, and especially when this is PEG, but this greater increase in stability is not proportional to the number of chains introduced. Therefore, although between 1 and 72 chains can be introduced, free amino groups can be left if subsequent derivations are desired or simply to reduce manufacturing costs. Preferably it they incorporate on average between 3 and 10 PEG chains per ferritin and more preferably between 4 and 6.
  • PEG derivatives useful for the present invention are detailed below: i) MeO-PEG-COOH: PEG1 156 MeO-PEG (1 1) -COOH a-Methoxy-oo-propanoic acid unde (ethylene glycol) PEG-WM 588.7 g / mol, PEG1 161 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 750 D, PEG1 158 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 2,000 D, PEG1 1 60 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 5,000 D, PEG1 157 MeO-PEG-COOH a-Methoxy- ⁇ - poly carboxylic acid (ethylene glycol) PEG-WM 10,000 D, PEG1 159 MeO-PEG (1 1)
  • succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton was preferably chosen.
  • the PEG derivative containing the succinimidyl ester functional group allows direct functionalization of the ferritins since this activated ester group reacts with the amino groups surrounding the nanoparticles, forming an amide-type covalent bond.
  • Experimental data has shown that after 3 h of injection, the particles accumulate significantly in the lung.
  • the amount of Tc / particle can be controlled (in a range 0-20, with 100% practically incorporating Te), it allows the optimum range of concentrations of Te (10 " ) to be exceeded. 9 M) and that of MRI (of the order of 10 "5 M).
  • the inner cavity of the ferritin can be used to introduce magnetite, obtaining a superparamagnetic nanoparticle called magnetoferritin, from now on the magnetoferritin of the invention.
  • This superparamagnetic nanoparticle is constituted by a magnetite nanoparticle encapsulated in ferritin that has properties to behave as a contrast agent in MRI.
  • the magnetoferritin of the invention is biocompatible and has a biodistribution in different organs, significantly improving the properties of other contrast agents such as magnetite alone. This can be used as a bimodal contrast agent in MRI.
  • the preparation of these nanoparticles can be occluded 99m Tc.
  • this is introduced in the form of 99m TcO 4 " .
  • the introduction of 99m TcO 4 can be carried out at room temperature at an optimal time for injection into the body.
  • it does not require reduction of Tc (VII) and its incorporation into the nanoparticle is practically total, which allows an optimal accumulation of the radionuclide.
  • the fact that the stircnectate is occluded in the Fe ore network makes it possible to control the amount of 99m TcO 4 " per particle and therefore, can Preparing drugs of different doses of radionuclide, depending on the needs.
  • the magnetoferritins of the invention can be used as a trimodal contrast agent, OI-MRI-SPECT.
  • the preferred Te species for inclusion in the magnetoferritin of the invention is 99m TcO 4 " .
  • the concentration of 99m Tc can be adjusted according to need, for example diagnosis or therapy but preferably is between 10 " 9 -10 " 5 M.
  • the present inventors have managed to obtain magnetite particles of high Fe content in the ferritin cavity.
  • the Fe content can be modulated up to 3800 Fe atoms per unit of ferritin
  • the amount of Fe from 3000 to 3800 Fe atoms per unit of ferritin, which improves its properties as a contrast agent in MRI.
  • the Te is occluded within the magnetite, which is an advantage over the complexes of coordination of Te since the concentration of Te per particle is much higher (up to 20 times higher) and allows a greater density of Te atoms to accumulate. This greater accumulation results in an increase in gamma radiation and a higher resolution. Also, the accumulation optimizes the concentration of the radiolabel, which allows the use of lower doses.
  • Another aspect is to provide a method for the synthesis of ferritins and magnetoferritins of the present invention. This method comprises: a) anchoring at least one quantum dot to the surface of the ferritin b) and then anchoring the PEG chains to the surface of the ferritin.
  • step (a) comprises: adding a solution comprising a quantum dot derivatized with carboxylic groups on the surface and a coupling agent, such as a carboimide; to another solution of the ferritin and subsequently reacting the solution comprising the PEG.
  • a coupling agent such as a carboimide
  • the introduction of biocompatible polymer chains must be carried out after the introduction of the QD.
  • the introduction of the biocompatible polymer is preferably carried out by using biocompatible polymer derivatives comprising carboxylic acids, and thus bonds are formed. amide type, so a coupling agent can be used for catalysis of the reaction.
  • the biopolymer may be activated / functionalized, that is, the carboxylic acids may be derivatized, including the coupling agent.
  • Preferred coupling agents of the present invention are selected from N, N-dicyclohexylcarbodiimide, 1 - [3- (dimethylamino) propyl] -3-ethylcarbodiimide, diisopropylcarbodiimide and any combination thereof.
  • this is done before the introduction of the QD and the biocompatible polymer.
  • the introduction of magnetite can be done through the process described by Douglas (Masaki Uchida, Masahiro Terashima, Charles H. Cunningham, Yoriyasu Suzuki, Deborah A. Willits, Ann F. Willis, Philip C. Yang, Philip S. Tsao, Michael V. McConnell, Mark J.
  • the method of the invention comprises at least the steps of: i) preparing an apoferritin solution, preferably of a concentration between 0.1 and 100 mg / mL, and more preferably between 5 and 20 mg / mL, ii) Preparing a solution that comprise Fe (ll) and Fe (lll) in stoichiometry of approximately 1: 2, that is, between 1: 1, 8 to 1, 8: 1 iii) prepare a solution of shovecnectate ( 99m Tc) iv) add the interleaved solution prepared in step (ii) and the solution prepared in step (iii) over that prepared in step (i).
  • the superparamagnetic magnetoferritin doped with 99m Tc is isolated
  • the initial stoichiometry of Fe (ll) and Fe (lll) is decisive for the proper preparation of magnetoferritin and hence its magnetic properties. Unlike the method reported by Douglas et al., Where all the initial Fe is in its oxidation state Fe (ll) and it has been proven that with a strict control of the extent of oxidation, in our method, the stoichiometric balance of departure confers optimal conditions and does not require exhaustive control of the air inlet in the system.
  • the iron (II) and (III) salts useful for the preparation of magnetoferritin are known to those skilled in the art.
  • step (ii) is prepared by mixing a solution comprising iron (II) sulfate ammonia hexahydrate with another comprising Fe (N0 3 ) 3 in HCI.
  • the inclusion of 99m Tc0 4 " is carried out by small additions of 99m Tc0 4" at the same time as the magnetite network is made.
  • the resulting solution can be dialyzed into dialysis bags with adequate pore size and separate particles from all remaining material.
  • the concentration of Te can be measured and in this way the percentage of Te incorporated is known.
  • the behavior of Mo0 4 2 " has been studied, because it has a chemistry very similar to 99m Tc0 4 " , but it is not radioactive. It has been observed that for small Mo contents (0-20 Mo / magnetoferritin atoms), the incorporation is practically 100%.
  • This technique allows us to directly study the electronic transitions that occur in the atom when it is subjected to a beam of high-energy electrons, 200kV.
  • this technique measures the energy that the incident electron loses when it interacts with an atom.
  • the L2.3 transitions are directly studied where the 2p electrons of the atom are transferred to unoccupied states above the Fermi level.
  • the energy required for this transition is a characteristic value for each atom and is equal to the energy lost by the incident electron.
  • V 513eV, O 532eV, Fe 708eV the different elements present in the nanoparticles
  • the fine structure of the EELS spectrum of transition metals is characterized by the presence of two intense peaks (white lines) whose intensity and position in energy varies depending on the oxidation state of the material (Leapman et al Phys. Rev. Lett. 45, 397 (1980), Turquat et al. International Journal of Inorganic Materials 3 (2001) 1025-1032).
  • the detailed study of the fine structure of the absorption peak reflects information on the electronic state of the material and can study the possible variations in the oxidation state of iron or vanadium through the nanoparticles.
  • an EELS spectrum was acquired, with dispersion energy of 0.5eV, every 0.6 nm along a 36.7nm line that passes through the nanoparticles.
  • the analysis of each of the acquired spectra (quantification and study of the oxidation state) along the nanoparticle gives us the composition and oxidation state of the metal characterized to the sub-nanometer scale.
  • step (a) it is buffered with AMPSO at a pH between pH 7.5 and 9.5, preferably between 8.0 and 9.0.
  • the chains of the biocompatible polymer, preferably PEG are reacted with the magnetoferritin after the inclusion of the magnetite / Tc.
  • Another aspect relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the magnetoferritins of the present invention and at least one pharmaceutically acceptable excipient, as well as the use of said pharmaceutical composition for the preparation of a medicament.
  • the ferritin of the present invention, and especially the magnetoferritin, as well as the pharmaceutical composition that includes them, are useful for the preparation of medicament for the diagnosis of different diseases, according to the use of molecules that confer specificity for a tissue or organ in question.
  • cancer including cervical, head and neck, renal and ureter, colon, rectum and anus, endometrial, esophagus, stomach, liver, larynx, ovarian, pancreas, skin cancer , prostate, lung, brain, testis, leukemia, melanoma, and lymphoma.
  • both the ferritin of the present invention, and especially magnetoferritin, as well as the pharmaceutical composition comprising them as contrast agents in general and as a contrast agent in MRI, OI or SPECT are also useful.
  • Figure 1 shows one of the particular embodiments of the nanostructure of the invention.
  • This consists of ferritin (i), with a magnetite core doped with 99m Tc (iv), a quantum dot anchored to the surface (ii), and biocompatible PEG polymer (iii).
  • the nanostructure is capable of acting as a contrast agent in MRI (i), OI (ii), scintigraphy (iv) and shows life times medium in blood sufficiently extensive to be distributed through the circulatory system without being phagocyted in a time less than 3h.
  • Figure 3 EELS in line of a ferritin-QD dimer, demonstrating the presence of Fe and O in the ferritin block and Cd in that of QD.
  • Figure 4. Image of athletic ratancillos to which the nanostructure object of the present patent was injected. After 3 h, the fluorescence of the nanostructure, preferentially centered in the lungs, is observed, with a practically negligible concentration in the liver. Examples
  • ferritin was coupled with 3 different types of quantum dots: a first type of quantum dots emitting in the green (QD525), a second type emitting in the red (QD655) and a third emitting in the near infrared (QD800).
  • QD525) a first type of quantum dots emitting in the green
  • QD655 a second type emitting in the red
  • QD800 a third emitting in the near infrared
  • EDC catalyst 1-ethyl-3- [3- dimethylaminopropyl] carbodiimide
  • the study by Optical Imaging of the biodistribution of magnetoferritin coupled to the QD800 showed the following: in a time between 15 minutes and 1 hour after intravenous administration the nanostructure is concentrated in the liver, lung and brain, in decreasing order. After 3 hours, it preferably located in the lungs, the concentration in the liver being very insignificant (Figure 4). After 24 hours no nanoparticles were detected.
  • Dissolution 1 A solution of apoferritin (Sigma-Aldrich Ref. A341 -1 G, lot. 048K7004) with a concentration of 10mg / ml in AMPSO buffer pH 8.6 (Sigma A6659) is prepared 10 ml. The solution is degassed with a strong stream of argon and under stirring for 10 min.
  • Solution 3 A solution of 0.1 M NaOH is degassed with a strong stream of argon and under stirring for 10 min.
  • Solution 4 A solution of a particulate ( 99m Tc) obtained from a commercial kit is degassed with a strong stream of argon and under stirring for 2 min.
  • Solution 6 Suspension 5 is slowly added 1 ml of a 0.1 M sodium citrate solution to remove all metal compounds that have not been encapsulated in apoferritin. The resulting solution is chromatographed (10 min) in size exclusion column (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17- 0851 -01), obtaining the final solution 6 containing mangnetite doped with 99m Tc encapsulated in the apoferritin cavity.
  • size exclusion column Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17- 0851 -01
  • Quantum dots QD525, QD655 and QD800 were purchased from Invitrogen (Q21341 MP, Q21321 MP, Q21371 MP, respectively).
  • the quantum dots are coated by a polymer functionalized with carboxylic groups to react with the amino groups of the ferritin.
  • the carboxylic residues of the QDs must be activated with EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide, Fluka 03450-25G).
  • Solution 7. 20 ⁇ of the commercial QD stock solution (8 mM) is incubated with 10 ⁇ of a stock EDC solution (10 mg / ml in double distilled water) for 30 min for activation. This preparation is carried out while degassing the solutions required in the previous step of magnetoferritin synthesis.
  • Solution 8 0.2 ml of solution 6 (magnetoferritin- 99m Tc) is diluted to 2 ml in PBS buffer (monobasic potassium phosphate, Acros Organics 424205000 and dibasic sodium phosphate, Acros Organics 424375000) and solution 7 is added ( quantum dot activated The mixture is incubated for 1 h under gentle agitation at 4 Q C. Subsequently the sample is purified on a chromatography column (15 min) by size exclusion (Sephacryl 5.5 cm x 1.5 cm bed , Sigma) in order to eliminate excess product that had not reacted.
  • PBS buffer monobasic potassium phosphate, Acros Organics 424205000 and dibasic sodium phosphate, Acros Organics 424375000
  • solution 7 quantum dot activated
  • the mixture is incubated for 1 h under gentle agitation at 4 Q C. Subsequently the sample is purified on a chromatography column (15 min) by size exclusion (Sephacryl
  • the polyethylene glycol derivative MeO-PEG-NHS a-Methoxy-Qj-carboxylic acid succinimidyl ester poly (ethylene glycol) (PEG-MW 2,000 Dalton) / M.W. 2000 g / mol) was purchased from Iris Biotech GmbH (PEG1 1 64, lot. 125447).
  • Solution 9 1000 moles of PEG (0.0058 g in 0.5 ml of double distilled water) were added to solution 8 and left 30 min under gentle stirring and at room temperature. Chromatograph (10 min) on a size exclusion column (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851 -01) until a pure solution of magnetite nanoparticles doped with 99m is obtained Tc, covalently coupled with a QD and PEG.
  • PEG 0.0058 g in 0.5 ml of double distilled water
  • Quantification of anchored quantum dot Only when the preparation is carried out in a strong excess of QD, can nanoparticles of the QD-magnetoferritin-QD type be observed.
  • the count of the number of QD convalently linked to the magnetoferritin in the nanostructure object of the present invention was carried out by TEM (Transmission Electron Microscopy) ( Figure 2) and by Scanning-Transmission Electron Microscopy in High-Angle Dark Field Mode Angle (HAADF-STEM) (figure 2).
  • HAADF-STEM Scanning-Transmission Electron Microscopy in High-Angle Dark Field Mode Angle
  • the relationship between the intensity in the image and the value of the atomic number is approximately of the Z 2 type (SJ Pennycook, DE Jesson, Ultramicroscopy, 1991, 37, 14).
  • the particles that show the lowest intensity would be the one that contains lighter elements (Fe, O) and the brightest is the one that contains the heaviest elements (Cd, Se).
  • EELS Energy loss spectroscopy of electrons. This technique, which is carried out in the transmission electron microscopes, records the kinetic energy that the incident electron beam loses as it passes through the sample. This loss of energy corresponds to the electron-sample inelastic interactions.

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Abstract

The invention relates to multifunctional nanostructures consisting of a ferritin characterised in that it comprises at least one quantum dot and at least one molecule of a biocompatible polymer, both of which are covalently bound to the surface of the ferritin. The invention also relates to a method for obtaining said nanostructures, and to the use thereof as a drug, preferably for the diagnosis of cancer or as a contrast agent.

Description

NANOESTRUCTURAS MU LTI FUNCIONALES COMO AGENTES DE DIAGNOSIS TRIMODAL MRI-OI-SPECT  MU LTI FUNCTIONAL NANOESTRUCTURES AS MRI-OI-SPECT TRIMODAL DIAGNOSIS AGENTS
La presente invención se refiere a una nanoestructura multifuncional, concretamente a una ferritina, además de su aplicación como agente de contraste en OI, MRI o SPECT. Por tanto, la invención se podría encuadrar dentro del campo de la biomedicina. The present invention relates to a multifunctional nanostructure, specifically a ferritin, in addition to its application as a contrast agent in OI, MRI or SPECT. Therefore, the invention could be framed within the field of biomedicine.
ESTADO DE LA TÉCNICA STATE OF THE TECHNIQUE
La integración de la Nanotecnología en la biotecnología ha hecho florecer una nueva disciplina: la nanomedicina. En este campo, se diseñan y preparan nanopartículas metálicas para obtener bioimágenes mediante el uso simultáneo de varias técnicas, distribución efectiva de fármacos o técnicas de terapias tan prometedoras como la hipertermia originada localmente por nanopartículas magnéticas. Es un área de tremendo potencial sujeta al desarrollo de nuevas nanoestructuras para su avance. The integration of Nanotechnology in biotechnology has made a new discipline flourish: nanomedicine. In this field, metal nanoparticles are designed and prepared to obtain bio-images through the simultaneous use of several techniques, effective distribution of drugs or techniques of promising therapies such as hyperthermia caused locally by magnetic nanoparticles. It is an area of tremendous potential subject to the development of new nanostructures for its advancement.
Las nanopartículas magnéticas han atraído atención principalmente por su uso potencial como agentes de contraste en Imagen por Resonancia Magnética (MRI). Esta técnica se basa en la resonancia magnética de los protones de tejidos del cuerpo (agua, membranas, lípidos, proteínas, etc) y es actualmente el método más potente de diagnosis. Magnetic nanoparticles have attracted attention primarily because of their potential use as contrast agents in Magnetic Resonance Imaging (MRI). This technique is based on the magnetic resonance of the protons of body tissues (water, membranes, lipids, proteins, etc.) and is currently the most powerful method of diagnosis.
El contraste en MRI, puede mejorarse con sustancias paramagnéticas. La capacidad de un compuesto para incrementar la velocidad de relajación de los espines de protón de las moléculas de agua del entorno se llama relajación y se define como R1 -1/T1 o R2-1 /T2. Las nanopartículas superparamagnéticas son candidatos para actuar como agentes de contraste en MRI. Al igual que las sustancias paramagnéticas, pierden su magnetización cuando se elimina el campo magnético externo, pero a diferencia de éstas, su magnetización es sensiblemente mayor. Por lo tanto, la relajación que producen es mucho más alta que las de los clásicos complejos paramagnéticos de Gd(lll). El efecto de las nanopartículas superparamagnéticas se puede describir en base a la heterogeneidad del intenso campo magnético que afecta a los protones del alrededor, induciendo un desfase del momento magnético y dando lugar a un acortamiento del tiempo de relajación T2. De este modo, las nanopartículas superparamagnéticas son unos buenos candidatos para el desarrollo de nuevos y elegantes agentes de contraste, permitiendo una detección temprana de patologías severas y de gran impacto social. Por otro lado, los denominados quantum dots (QDs), han sido usados con éxito como nuevos marcadores fluorescentes en el campo biomédico y son considerados como una herramienta prometedora en Imagen óptica de fluorescencia (OI) para diagnóstico clínico. Los QDs son nanopartículas inorgánicas, generalmente compuestas de elementos de los grupos ll-VI y lll-V, los cuales, debido a su confinamiento cuántico de cargas en un diminuto espacio muestran unas propiedades fluorescentes únicas: espectros de emisión estrechos, rendimiento cuántico alto, espectros de absorción anchos, buena estabilidad química y alta fotoestabilidad y longitud de onda de emisión dependiente del tamaño, ampliando su rango de emisión hasta la región NIR (infrarrojo cercano) o IR (infrarrojo) y ofreciendo una mayor penetración en tejido para una mejor imagen. Sin embargo, a pesar de las excepcionales propiedades fluorescentes que presentan, estas nanopartículas necesitan ser funcionalizadas con algún tipo de polímero o proteína que las haga biocompatibles y por tanto aptas para su utilización in vivo. The contrast in MRI can be improved with paramagnetic substances. The ability of a compound to increase the relaxation rate of proton spins of surrounding water molecules is called relaxation and is defined as R1 -1 / T1 or R2-1 / T2. Superparamagnetic nanoparticles are candidates to act as contrast agents in MRI. Like paramagnetic substances, they lose their magnetization when the external magnetic field is removed, but unlike them, their magnetization is significantly higher. Therefore, the relaxation they produce is much more high than those of the classic paramagnetic complexes of Gd (lll). The effect of superparamagnetic nanoparticles can be described based on the heterogeneity of the intense magnetic field that affects the surrounding protons, inducing a lag of the magnetic moment and resulting in a shortening of the relaxation time T2. Thus, superparamagnetic nanoparticles are good candidates for the development of new and elegant contrast agents, allowing early detection of severe pathologies and great social impact. On the other hand, the so-called quantum dots (QDs), have been used successfully as new fluorescent markers in the biomedical field and are considered as a promising tool in Fluorescence Optical Imaging (OI) for clinical diagnosis. The QDs are inorganic nanoparticles, generally composed of elements of groups ll-VI and lll-V, which, due to their quantum confinement of charges in a tiny space show unique fluorescent properties: narrow emission spectra, high quantum yield, Wide absorption spectra, good chemical stability and high photostability and emission wavelength dependent on size, extending its emission range to the NIR (near infrared) or IR (infrared) region and offering greater tissue penetration for a better image . However, despite the exceptional fluorescent properties they present, these nanoparticles need to be functionalized with some type of polymer or protein that makes them biocompatible and therefore suitable for use in vivo.
Un enfoque dentro de la nanomedicina es el uso de nanopartículas que puedan combinar diferentes técnicas de bioimagen. Cada modalidad de bioimagen tiene sus propios méritos pero también ciertas desventajas y por lo tanto los métodos de imagen multimodales presentan mayor capacidad para obtener una imagen integral y más detallada. La combinación MRI-OI es un buen ejemplo de un método biomodal y una ruta para su consecución es el uso de nanoestructuras que contengan dos componentes metálicos, uno magnético y otro fluorescente: nanopartículas bifuncionales magneto-fluorescente. El nanocomponente magnético puede incorporar un radiomarcador, como 99mTc04 ", añadiendo a la nanoestructura multifuncional una nueva modalidad de imagen médica mediante la detección de la radiación gamma que dicho radionúclido genera mediante gammagrafía (SPECT). One approach within nanomedicine is the use of nanoparticles that can combine different bioimaging techniques. Each bioimaging modality has its own merits but also certain disadvantages and therefore multimodal imaging methods have a greater capacity to obtain a comprehensive and more detailed image. The MRI-OI combination is a good example of a biomodal method and a route for its achievement is the use of nanostructures that contain two metallic components, one magnetic and other fluorescent: magneto-fluorescent bifunctional nanoparticles. The magnetic nanocomponent can incorporate a radiolabel, such as 99m Tc0 4 " , adding a new medical imaging modality to the multifunctional nanostructure by detecting the gamma radiation that the radionuclide generates by scintigraphy (SPECT).
Un punto clave para la aplicación biomédica de las nanopartículas metálicas es que no pueden ser tóxicas y deben permanecer en circulación el tiempo suficiente para alcanzar el blanco biológico. El sistema fagocítico mononuclear (MPS) reconoce y elimina, a través de los macrófagos, las partículas de la circulación, con su simultánea concentración en órganos con alta actividad fagocítica (principalmente el hígado). Por lo tanto, una estrategia sensata es el uso de nanopartículas que sean capaces de evadir el ataque por parte del MPS y no sean fagocitadas por los macrófagos, aumentando consecuentemente el tiempo de vida media en plasma y permitiendo alcanzar un órgano o tejido específico. A key point for the biomedical application of metal nanoparticles is that they cannot be toxic and must remain in circulation long enough to reach the biological target. The mononuclear phagocytic system (MPS) recognizes and eliminates, through macrophages, the particles of the circulation, with their simultaneous concentration in organs with high phagocytic activity (mainly the liver). Therefore, a sensible strategy is the use of nanoparticles that are able to evade the attack by the MPS and are not phagocytosed by macrophages, consequently increasing the half-life in plasma and allowing to reach a specific organ or tissue.
Este tipo de agentes de diagnosis ideales para su aplicación clínica deberían poseer una alta y específica acumulación en las células adecuadas, lo que tendría como consecuencia la posibilidad de diagnosticar de forma cada vez más precoz una enfermedad aún tratable. This type of diagnostic agents ideal for clinical application should have a high and specific accumulation in the appropriate cells, which would result in the possibility of diagnosing an even treatable disease more and more early.
Numerosos métodos físicos y químicos han sido utilizados para preparar nanopartículas magnéticas. Puesto que las propiedades magnéticas son muy dependientes del tamaño, es crucial que el método a desarrollar permita la obtención de nanopartículas con tamaños uniformes. Una posible ruta para obtener nanopartículas metálicas sin agregación y con tamaño controlado es el uso de una plataforma molecular preorganizada, con una cavidad que pueda actuar como nanoreactor para el control químico y espacial en la formación de las nanopartículas. Un ejemplo típico de este tipo de moléculas es la proteína ferritina. La apoferritina consiste en una proteína esférica formada por 24 subunidades rodeando una cavidad acuosa con un diámetro de aproximadamente 8 nm. Numerous physical and chemical methods have been used to prepare magnetic nanoparticles. Since the magnetic properties are very dependent on size, it is crucial that the method to be developed allows the obtaining of nanoparticles with uniform sizes. A possible route to obtain metallic nanoparticles without aggregation and with controlled size is the use of a pre-organized molecular platform, with a cavity that can act as a nanoreactor for chemical and spatial control in the formation of nanoparticles. A typical example of this type of molecules is the ferritin protein. Apoferritin consists of a spherical protein formed by 24 subunits surrounding an aqueous cavity with a diameter of approximately 8 nm.
La organización de las multisubunidades para formar la apoferritina genera la presencia de canales. Ocho canales hidrofílicos de aproximadamente 4 Á permiten la entrada de iones metálicos y moléculas suficientemente pequeñas al interior de la cavidad de la proteína. Esto ha permitido la introducción de magnetita en el interior de la apoferritina produciendo magnetoferritina, la cual ha sido usada como método de diagnostico monomodal (MRI) (Journal of Magnetic Resonance Imaging. 4(3):497-505, 1994 May-Jun.). The organization of multisubunities to form apoferritin generates the presence of channels. Eight hydrophilic channels of approximately 4A allow the entry of sufficiently small metal ions and molecules into the protein cavity. This has allowed the introduction of magnetite into the apoferritin producing magnetoferritin, which has been used as a single-mode diagnostic method (MRI) (Journal of Magnetic Resonance Imaging. 4 (3): 497-505, 1994 May-Jun. ).
DESCRIPCIÓN DETALLADA DE LA INVENCIÓN DETAILED DESCRIPTION OF THE INVENTION
En vista del estado de la técnica un objetivo de la presente invención es proporcionar un agente de contrate OI, el cual puede además se puede ser usado como agente bimodal, OI-MRI, e incluso trimodal, OI-MRI-SPECT, basado en una ferritina, caracterizada porque comprende al menos un quantum dot (QD) y al menos una molécula de un polímero biocompatible unidos ambos covalentemente a la superficie de la ferritina (Figura 1 ). Estas partículas han demostrado que se en diferentes tejidos, como el hígado, pulmón y cerebro, por lo que son útiles como agentes de contraste para enfermedades en duchos tejidos. In view of the state of the art, an objective of the present invention is to provide an OI contracting agent, which can also be used as a bimodal agent, OI-MRI, and even trimodal, OI-MRI-SPECT, based on a ferritin, characterized in that it comprises at least one quantum dot (QD) and at least one molecule of a biocompatible polymer both covalently bonded to the surface of the ferritin (Figure 1). These particles have been shown to be in different tissues, such as the liver, lung and brain, so they are useful as contrast agents for diseases in tissue tissues.
La ferritina de la invención es biocompatible y sorprendentemente la bioconjugación con un quantum dot, mejora significativamente la biodistribución de ambas partículas por separado. La nanoestructuratura formada por la conjugación de la ferritina y del quantum dot puede ser usada como agente de contraste en OI y muestra tiempos de vida medios suficientemente extensos para distribuirse por el sistema circulatorio sin ser fagocitados en un tiempo menor de 3h, pero a la vez suficientemente cortos para evitar su acumulación en el cuerpo. En el contexto de la presente invención se entiende por ferritina cualquier apo- ferritina, independiente del material que se encuentre encapsulado en su cavidad interna, incluidas las apo-ferritinas las cuales la cavidad interna está vacía. The ferritin of the invention is biocompatible and surprisingly bioconjugate with a quantum dot, significantly improves the biodistribution of both particles separately. The nanostructure formed by the conjugation of ferritin and quantum dot can be used as a contrast agent in RO and shows sufficiently long lifespan to be distributed through the circulatory system without being phagocyted in less than 3 hours, but at the same time Short enough to prevent its accumulation in the body. In the context of the present invention, ferritin is understood as any apophritin, independent of the material that is encapsulated in its internal cavity, including the apo-ferritins which the internal cavity is empty.
Los componentes fluorescentes, como por ejemplo los QD525, QD655 y QD800, que podría enlazar covalentemente a la capa externa de la ferritina mediante formación de un enlace covalente amida por reacción entre los grupos -NH2 libres de los restos de lisinas de la capa externa de la ferritina y los grupos carboxílicos del QD. The fluorescent components, such as QD525, QD655 and QD800, which could covalently bind to the outer layer of the ferritin by forming a covalent amide bond by reaction between the -NH 2 groups free of the lysine residues of the outer layer of ferritin and the carboxylic groups of QD.
Los denominados quantum dots QDs, han sido usados con éxito como nuevos marcadores fluorescentes en el campo biomédico y son considerados como una herramienta prometedora en Optical Imaging para diagnóstico clínico in vivo [Choi, H. _S. Liu, W. Misra, W., Tanaka, E., Zimmer, J. P., Ipe, B. I., Bawendi, M. G., Frangioni, J. V., Nal Biotechnol. 2007, 25, 1165-1170 and Qi, L, Gao, X. Expert. Opin. Drug Delivery 2008, 5, 263-267.]. Los QDs son nanopartículas inorgánicas, generalmente compuestas de elementos de los grupos ll-VI y lll-V, los cuales, debido a su confinamiento cuántico de cargas en un diminuto espacio muestran unas propiedades fluorescentes únicas: espectros de emisión estrechos, rendimiento cuántico alto, espectros de absorción anchos, buena estabilidad química y alta fotoestabilidad y longitud de onda de emisión dependiente del tamaño, ampliando su rango de emisión y ofreciendo una mayor penetración en tejido para una mejor imagen. Una de las limitaciones de los QD para poder tener aplicaciones in vivo es su bajo tiempo de residencia en sangre. No obstante, esta limitación puede ser superada mediante funcionalización con polímeros hidrofóbicos como el polietilén glicol (PEG) [Daou, T. Jean; Li, Liang; Reiss, Peter; Josserand, Veronique; Texier, Isabelle . Langmuir (2009), 25(5), 3040-3044 y referencias]. Una vez convenientemente funcionalizados, pueden ser administrados in vivo y su localización detectada por la emisión fluorescente correspondiente. Los quantum dots se pueden seleccionar por ejemplo entre los que se listan en el catálogo Invitrogen Molecular Probes 2009 y preferiblemente se seleccionan entre Qdot® 625 ITK™ carboxyl quantum dots (A1 0200), Qdot® 605 ITK™ carboxyl quantum dots 8 μΜ solution (Q21301 MP), Qdot® 585 ITK™ carboxyl quantum dots 8 μΜ solution (Q21 31 1 MP), Qdot® 655 ITK™ carboxyl quantum dots 8 μΜ solution (Q21 321 MP), Qdot® 565 ITK™ carboxyl quantum dots 8 μΜ solution (Q21 331 MP), Qdot® 525 ITK™ carboxyl quantum dots 8 μΜ solution (Q21 341 MP), Qdot® 705 ITK™ carboxyl quantum dots 8 μΜ solution (Q21 361 MP), Qdot® 800 ITK™ carboxyl quantum dots 8 μΜ solution (Q21 371 MP), Qdot® 545 ITK™ carboxyl quantum dots 8 μΜ solution (Q21 391 MP). Qdot® 605 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 501 MP), Qdot® 585 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 51 1 MP), Qdot® 655 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 521 MP), Qdot® 565 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 531 MP), Qdot® 525 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 541 MP), Qdot® 705 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 561 MP), Qdot® 800 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 571 MP), Qdot® 545 ITK™ amino (PEG) quantum dots 8 μΜ solution (Q21 591 MP) y cualquiera de sus combinaciones. Los quantum dots preferidos son los que emiten entre 750 y 850 nm. The so-called quantum dots QDs, have been used successfully as new fluorescent markers in the biomedical field and are considered as a promising tool in Optical Imaging for in vivo clinical diagnosis [Choi, H. _S. Liu, W. Misra, W., Tanaka, E., Zimmer, JP, Ipe, BI, Bawendi, MG, Frangioni, JV, Nal Biotechnol. 2007, 25, 1165-1170 and Qi, L, Gao, X. Expert. Opin. Drug Delivery 2008, 5, 263-267.]. The QDs are inorganic nanoparticles, generally composed of elements of groups ll-VI and lll-V, which, due to their quantum confinement of charges in a tiny space show unique fluorescent properties: narrow emission spectra, high quantum yield, Wide absorption spectra, good chemical stability and high photostability and emission wavelength dependent on size, expanding its emission range and offering greater tissue penetration for a better image. One of the limitations of the QD to be able to have applications in vivo is their low residence time in blood. However, this limitation can be overcome by functionalization with hydrophobic polymers such as polyethylene glycol (PEG) [Daou, T. Jean; Li, Liang; Reiss, Peter; Josserand, Veronique; Texier, Isabelle. Langmuir (2009), 25 (5), 3040-3044 and references]. Once conveniently functionalized, they can be administered in vivo and their location detected by the corresponding fluorescent emission. The quantum dots can be selected, for example, from those listed in the Invitrogen Molecular Probes 2009 catalog and preferably are selected from Qdot® 625 ITK ™ carboxyl quantum dots (A1 0200), Qdot® 605 ITK ™ carboxyl quantum dots 8 μΜ solution ( Q21301 MP), Qdot® 585 ITK ™ carboxyl quantum dots 8 μΜ solution (Q21 31 1 MP), Qdot® 655 ITK ™ carboxyl quantum dots 8 μΜ solution (Q21 321 MP), Qdot® 565 ITK ™ carboxyl quantum dots 8 μΜ solution (Q21 331 MP), Qdot® 525 ITK ™ carboxyl quantum dots 8 μΜ solution (Q21 341 MP), Qdot® 705 ITK ™ carboxyl quantum dots 8 μΜ solution (Q21 361 MP), Qdot® 800 ITK ™ carboxyl quantum dots 8 μΜ solution (Q21 371 MP), Qdot® 545 ITK ™ carboxyl quantum dots 8 μΜ solution (Q21 391 MP). Qdot® 605 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 501 MP), Qdot® 585 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 51 1 MP), Qdot® 655 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 521 MP), Qdot® 565 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 531 MP), Qdot® 525 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 541 MP) , Qdot® 705 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 561 MP), Qdot® 800 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 571 MP), Qdot® 545 ITK ™ amino (PEG) quantum dots 8 μΜ solution (Q21 591 MP) and any of its combinations. Preferred quantum dots are those that emit between 750 and 850 nm.
Aunque hay diferentes maneras de anclar el quantum dot a la superficie de la ferritina, preferiblemente estos están anclados a través de enlaces covalentes tipo amido, aprovechando los grupos amino que están libres en la superficie de la ferritina. Although there are different ways of anchoring the quantum dot to the surface of the ferritin, preferably these are anchored through covalent bonds type amido, taking advantage of the amino groups that are free on the surface of the ferritin.
Normalmente entre 1 y 2 unidades de quantum dots anclados a la superficie son suficientes para el uso de la ferritina como agente de contraste. Normally between 1 and 2 units of quantum dots anchored to the surface are sufficient for the use of ferritin as a contrast agent.
El número de quantum dots anclados a la magnetoferritina se puede calcular según la fórmula: N = 2n (RFE + RQD)2 / (3)1/2 (RQD)2, donde RFE es el radio de la nanopartícula y RQD es el radio del quantum dot (QD). Esta expresión, estimativa, proviene de dividir el área de las ferritina cubierta por los quantum dots y dividirla por el área de ferritina cubierta por un solo quantum dot. El cálculo asume un empaquetamiento compacto de QDs sobre la superficie de la ferritina y tiene en consideración los huecos entre los QDs. Por ejemplo para una partícula magnética de 10 nm el número máximo de QDs aumenta desde 45 hasta 133 cuando el diámetro del QD disminuye de 4 a 2 nm (D. Wang, J. He, N. Rosenzweig, Z. Rosenzweig, Nano Lett. 2004, 4, 409-413). The number of quantum dots anchored to the magnetoferritin can be calculated according to the formula: N = 2n (R FE + RQD) 2 / (3) 1/2 (RQD) 2 , where R FE is the radius of the nanoparticle and R QD It is the radius of the quantum dot (QD). This expression, estimated, it comes from dividing the area of the ferritin covered by the quantum dots and dividing it by the area of ferritin covered by a single quantum dot. The calculation assumes a compact packing of QDs on the surface of the ferritin and takes into account the gaps between the QDs. For example, for a magnetic particle of 10 nm the maximum number of QDs increases from 45 to 133 when the diameter of the QD decreases from 4 to 2 nm (D. Wang, J. He, N. Rosenzweig, Z. Rosenzweig, Nano Lett. 2004, 4, 409-413).
De acuerdo con el primer aspecto de la presente invención la ferritina tiene cadenas de un polímero biocompatible covalentemente unidas a la superficie de la ferritina. La presencia de este polímero mejora las propiedades de la ferritina de la invención para ser usada como agente de contraste puesto que aumenta su estabilidad general obteniendo tiempos medios de vida en sangre adecuados para su uso. El polímero biocompatible preferido es polietilénglicol (PEG), entre otras razones por su disponibilidad industrial, su facilidad de incorporación a la superficie de la ferritina y su alta biocompatibilidad. El proceso de unir covalentemente el polímero de PEG a otras moléculas, normalmente fármacos o proteínas terapéuticas es conocido como PEGylación (Kohler, N. ; Fryxell, G. E. ; Zhang, M. J. Am. Chem. Soc. 2004, 126, 7206. Paul, K. G. ; Frigo, T. B. ; Groman, J. Y. ; Groman, E. V. Bioconjugate Chem. 2004, 15, 394.). En nuestro caso, el PEG va unido covalentemente a la ferritina. Este proceso se puede llevar a cabo de forma sencilla, incubando un derivado del PEG reactivo con las nanopartículas. La unión covalente de PEG las enmascara del sistema inmune, aumenta la talla hidrodinámica (talla en solución) lo que aumenta el tiempo de vida media en sangre y reduce su eliminación por el sistema inmune. Este proceso además aumenta la solubilidad en agua de dichas nanopartículas y le confiere de forma general una estabilidad adicional. Se puede además controlar el número de moléculas de PEG que se unen a la superficie de la nanopartícula. Los derivados del PEG de tipo succinimidil ester permiten la formación de un enlace covalente de tipo amida por reacción con los grupos amino en la superficie externa de las nanopartículas. En resumen, para conseguir la unión de las cadenas de PEG y la ferritina, el PEG debe de estar derivatizado con grupos funcionales capaces de unirse por si solos o con la ayuda de un reactivo a la superficie de la ferritina. El PEG se une preferiblemente mediante enlaces amida, siendo por lo tanto los derivados de PEG preferidos, como ya se ha comentado antes, los que contienen el grupo funcional succinimidil ester, que permite una funcionalización directa de las ferritina, ya que este grupo éster activado reacciona con los grupos aminos que rodean las nanopartículas, formando el enlace covalente tipo amida. Uno de los PEG preferidos es PEG1 1 63 MeO- PEG-COO-Su a-Metoxi-cjo-ácido carboxílico succinimidil éster poli(etilenglicol) PEG-WM 2,000 Dalton. Los PEG funcionalizados como ester succinimidil son comerciales y su acoplamiento covalente a la partícula superparamagnético se puede llevar a cabo después de la introducción del 99mTc. In accordance with the first aspect of the present invention, ferritin has chains of a biocompatible polymer covalently bonded to the surface of ferritin. The presence of this polymer improves the properties of the ferritin of the invention to be used as a contrast agent since it increases its overall stability, obtaining adequate average life times in the blood for use. The preferred biocompatible polymer is polyethylene glycol (PEG), among other reasons for its industrial availability, its ease of incorporation to the surface of ferritin and its high biocompatibility. The process of covalently binding the PEG polymer to other molecules, usually drugs or therapeutic proteins, is known as PEGylation (Kohler, N.; Fryxell, GE; Zhang, MJ Am. Chem. Soc. 2004, 126, 7206. Paul, KG ; Frigo, TB; Groman, JY; Groman, EV Bioconjugate Chem. 2004, 15, 394.). In our case, PEG is covalently bound to ferritin. This process can be carried out easily, by incubating a derivative of the reactive PEG with the nanoparticles. The covalent union of PEG masks them of the immune system, increases hydrodynamic size (size in solution) which increases the half-life in blood and reduces their elimination by the immune system. This process also increases the water solubility of said nanoparticles and generally gives it additional stability. The number of PEG molecules that bind to the surface of the nanoparticle can also be controlled. PEG derivatives of the succinimidyl ester type allow the formation of a covalent bond of the amide type by reaction with the amino groups on the outer surface of the nanoparticles. In summary, to achieve the union of PEG chains and Ferritin, PEG must be derivatized with functional groups capable of binding on their own or with the aid of a reagent to the surface of ferritin. The PEG is preferably linked by amide bonds, therefore the preferred PEG derivatives, as mentioned above, contain the succinimidyl ester functional group, which allows a direct functionalization of the ferritin, since this activated ester group It reacts with the amino groups surrounding the nanoparticles, forming the covalent bond type amide. One of the preferred PEGs is PEG1 1 63 MeO-PEG-COO-Su a-Methoxy-cjo-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton. PEGs functionalized as succinimidyl ester are commercial and their covalent coupling to the superparamagnetic particle can be carried out after the introduction of 99m Tc.
El número de cadenas de PEG introducidas se pueden controlar dependiendo de la estequiometría y del PEG empleados. Por ejemplo, cuando se utilizan derivados de éster activados, dada la alta reactivad de dichos ésteres y de las aminas de la nanopartícula, la reacción es completa, de tal forma que el número de PEG enlazados covalentemente a la superficie de la nanopartícula coincide con el número de moléculas del derivado de PEG/nanopartículas que se utiliza en la reacción. Además el número de cadenas introducidas se comprobó a través del uso de un patrón electroforético, analizando las diferentes ferritinas derivatizadas con PEG y viendo su correspondencia con un aumento escalonado y gradual del peso molecular y por ende de una mayor retención. El PEG puede ser mono- o bifuncionalizado con ésteres activados y se pueden unir PEG con diferente densidad y cristalinidad. La estabilidad de la ferritina de la invención se ve aumentada por la introducción del polímero biocompatible, y en especial cuando este es PEG, pero este mayor aumento de la estabilidad no es proporcional al número de cadenas introducidas. Por lo tanto, aunque se puede introducir entre 1 y 72 cadenas, se pueden dejar grupos amino libre por si se desea realizar subsiguientes derivarizaciones o simplemente para reducir los coste de fabricación. Preferiblemente se incorporan de media entre 3 y 10 cadenas de PEG por ferritina y más preferiblemente entre 4 y 6. The number of PEG chains introduced can be controlled depending on the stoichiometry and PEG used. For example, when activated ester derivatives are used, given the high reactivity of said esters and the amines of the nanoparticle, the reaction is complete, such that the number of PEG covalently bonded to the surface of the nanoparticle coincides with the number of molecules of the PEG derivative / nanoparticles that is used in the reaction. In addition, the number of chains introduced was checked through the use of an electrophoretic pattern, analyzing the different ferritins derivatized with PEG and seeing their correspondence with a gradual and gradual increase in molecular weight and therefore a greater retention. The PEG can be mono- or bifunctionalized with activated esters and PEG can be joined with different density and crystallinity. The ferritin stability of the invention is enhanced by the introduction of the biocompatible polymer, and especially when this is PEG, but this greater increase in stability is not proportional to the number of chains introduced. Therefore, although between 1 and 72 chains can be introduced, free amino groups can be left if subsequent derivations are desired or simply to reduce manufacturing costs. Preferably it they incorporate on average between 3 and 10 PEG chains per ferritin and more preferably between 4 and 6.
A continuación se detallan algunos de los derivados de PEG útiles para la presente invención: i) MeO-PEG-COOH: PEG1 156 MeO-PEG(1 1 )-COOH a-Metoxi-oo-ácido propanoico unde(etilenglicol) PEG-WM 588,7 g/mol, PEG1 161 MeO-PEG- COOH a-Metoxi-oo-ácido carboxilico poli(etilenglicol) PEG-WM 750 D, PEG1 158 MeO-PEG-COOH a-Metoxi-oo-ácido carboxilico poli(etilen glicol) PEG-WM 2.000 D, PEG1 1 60 MeO-PEG-COOH a-Metoxi-oo-ácido carboxilico poli(etilenglicol) PEG-WM 5.000 D, PEG1 157 MeO-PEG-COOH a-Metoxi-ω- ácido carboxilico poli(etilenglicol) PEG-WM 10.000 D, PEG1 159 MeO-PEG- COOH a-Metoxi- -carboxílico ácido poli(etilenglicol) PEG-WM 20.000 D, PEG1 1 66 MeO-PEG-COO-Su a-Metoxi-oo-ácido carboxilico succinimidil éster poli(etilenglicol) PEG-WM 750 D, PEG1 1 63 MeO-PEG-COO-Su a-Metoxi-ω- carboxílico ácido succinimidil éster poli(etilenglicol) PEG-WM 2.000 D, PEG1 1 65 MeO-PEG-COO-Su a-Metoxi-oo-ácido carboxilico succinimidil éster poli(etilenglicol) PEG-WM 5.000 D, PEG1 162 MeO-PEG-COO-Su a-Metoxi-ω- ácido carboxilico succinimidil éster poli(etilenglicol) PEG-WM 10.000 D, PEG1 1 64 MeO-PEG-COO-Su a-Metoxi-oo-ácido carboxilico succinimidil éster poli(etilenglicol) PEG-WM 20.000 D, ii) HOOC-PEG-COOH:PEG1091 HOOC-PEG(12)-COOH a,ü>Bis(propionico ácido) duodeca(etilen glicol) PEG-WM 2.000 690,8 g/mol, PEG1083 HOOC-Some of the PEG derivatives useful for the present invention are detailed below: i) MeO-PEG-COOH: PEG1 156 MeO-PEG (1 1) -COOH a-Methoxy-oo-propanoic acid unde (ethylene glycol) PEG-WM 588.7 g / mol, PEG1 161 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 750 D, PEG1 158 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 2,000 D, PEG1 1 60 MeO-PEG-COOH a-Methoxy-oo-carboxylic acid poly (ethylene glycol) PEG-WM 5,000 D, PEG1 157 MeO-PEG-COOH a-Methoxy-ω- poly carboxylic acid (ethylene glycol) PEG-WM 10,000 D, PEG1 159 MeO-PEG-COOH a-Methoxy-carboxylic acid poly (ethylene glycol) PEG-WM 20,000 D, PEG1 1 66 MeO-PEG-COO-Su a-Methoxy-oo-acid carboxylic succinimidyl ester poly (ethylene glycol) PEG-WM 750 D, PEG1 1 63 MeO-PEG-COO-Su a-Methoxy-ω-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 D, PEG1 1 65 MeO-PEG- COO-Su a-Methoxy-oo-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 5,000 D, PEG1 162 Me O-PEG-COO-Su a-Methoxy-ω- carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 10,000 D, PEG1 1 64 MeO-PEG-COO-Su a-Methoxy-oo-carboxylic acid succinimidyl ester poly (ethylene glycol ) PEG-WM 20,000 D, ii) HOOC-PEG-COOH: PEG1091 HOOC-PEG (12) -COOH a, ü> Bis (propionic acid) duodeca (ethylene glycol) PEG-WM 2,000 690.8 g / mol, PEG1083 HOOC-
PEG-COOH α,ω-Bis-carboxi poli(etilen glicol) PEG-WM 2.000 D, PEG1085 HOOC-PEG-COOH α,ω-Bis-carboxi poli(etilen glicol) PEG-WM 3.000 DPEG1086 HOOC-PEG-COOH α,ω-Bis-carboxi poli(etilenglicol) PEG-WM 6.000 D, PEG1082 HOOC-PEG-COOH α,ω-Bis-carboxi poli(etilenglicol) PEG- WM 10.000 D PEG1084 HOOC-PEG-COOH α,ω-Bis-carboxi poli(etilen glicol) PEG-WM 20.000 D, iii) NHS-PEG-NHS:PEG1 184 Su-OOC-PEG-COO-Su α,ω-Di-succinimidil éster poli(etilen glicol) PEG-WM 2.000 D, PEG1 186 Su-OOC-PEG-COO-Su α,ω-Di- succinimidil éster poli(etilen glicol) PEG-WM 3.000 D, PEG1 187 Su-OOC-PEG- COO-Su α,ω-Di-succinimidil éster poli(etilen glicol) PEG-WM 6.000 D, PEG1 183 Su-OOC-PEG-COO-Su α,ω-Di-succinimidil éster poli(etilen glicol) PEG-WM 10.000 D, PEG1 185 Su-OOC-PEG-COO-Su α,ω-Di-succinimidil éster poli(etilen glicol) PEG-WM 20.000 D, iv).H2N-PEG-COOH:PEG1096 H2N-PEG- COOhTHCI a-Amino-Qj-carboxi poli(etilen glicol) clorohidrato PEG-WM 3.000 D, PEG1097 H2N-PEG-COOH*HCI a-Amino-oo-carboxi poli(etilenglicol) clorohidrato PEG-WM 5.000 Dalton PEG1095 H2N-PEG-COOH*HCI a-Amino- ω-carboxi poli(etilenglicol) clorohidrato PEG-WM 10.000 Dalton. PEG-COOH α, ω-Bis-carboxy poly (ethylene glycol) PEG-WM 2,000 D, PEG1085 HOOC-PEG-COOH α, ω-Bis-carboxy poly (ethylene glycol) PEG-WM 3,000 DPEG1086 HOOC-PEG-COOH α , ω-Bis-carboxy poly (ethylene glycol) PEG-WM 6,000 D, PEG1082 HOOC-PEG-COOH α, ω-Bis-carboxy poly (ethylene glycol) PEG- WM 10,000 D PEG1084 HOOC-PEG-COOH α, ω-Bis- carboxy poly (ethylene glycol) PEG-WM 20,000 D, iii) NHS-PEG-NHS: PEG1 184 Su-OOC-PEG-COO-Su α, ω-Di-succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 D, PEG1 186 Su-OOC-PEG-COO-Su α , ω-Di- succinimidyl ester poly (ethylene glycol) PEG-WM 3,000 D, PEG1 187 Su-OOC-PEG-COO-Su α, ω-Di-succinimidyl ester poly (ethylene glycol) PEG-WM 6,000 D, PEG1 183 Su-OOC-PEG-COO-Su α, ω-Di-succinimidyl poly (ethylene glycol) ester PEG-WM 10,000 D, PEG1 185 Su-OOC-PEG-COO-Su α, ω-Di-succinimidyl poly (ethylene) ester glycol) PEG-WM 20,000 D, iv) .H 2 N-PEG-COOH: PEG1096 H 2 N-PEG-COOhTHCI a-Amino-Qj-carboxy poly (ethylene glycol) hydrochloride PEG-WM 3,000 D, PEG1097 H 2 N -PEG-COOH * HCI a-Amino-oo-carboxy poly (ethylene glycol) PEG-WM hydrochloride 5,000 Dalton PEG1095 H 2 N-PEG-COOH * HCI a-Amino- ω-carboxy poly (ethylene glycol) PEG-WM 10,000 hydrochloride Dalton .
Se elegió preferiblemente el PEG1 1 63 MeO-PEG-COO-Su a-Metoxi-ω- carboxílico ácido succinimidil éster poli(etilen glicol) PEG-WM 2.000 Dalton. El derivado de PEG que contiene el grupo funcional succinimidil éster, permite una funcionalización directa de las ferritinas ya que este grupo éster activado reacciona con los grupos aminos que rodean las nanopartículas, formando un enlace covalente tipo amida. Los datos experimentales han demostrado que después de 3 h de inyección, las partículas se acumulan de forma significativa en pulmón. Por otra parte, puesto que se puede controlar la cantidad de Tc/partícula (en un rango 0-20, con un 100% prácticamente de incorporación de Te) permite que se pueda alcanzar de sobra el rango óptimo de concentraciones de Te (10"9 M) y el de MRI (del orden de 10"5 M). PEG1 1 63 MeO-PEG-COO-Su a-Methoxy-ω-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton was preferably chosen. The PEG derivative containing the succinimidyl ester functional group allows direct functionalization of the ferritins since this activated ester group reacts with the amino groups surrounding the nanoparticles, forming an amide-type covalent bond. Experimental data has shown that after 3 h of injection, the particles accumulate significantly in the lung. On the other hand, since the amount of Tc / particle can be controlled (in a range 0-20, with 100% practically incorporating Te), it allows the optimum range of concentrations of Te (10 " ) to be exceeded. 9 M) and that of MRI (of the order of 10 "5 M).
Se puede utilizar la cavidad interior de la ferritina para introducir magnetita, obteniendo una nanopartícula superparamagnética llamada magnetoferritina, a partir de ahora la magnetoferritina de la invención. Esta nanopartícula superparamagnética está constituida por una nanopartícula de magnetita encapsulada en la ferritina que presenta propiedades para comportarse como agente de contraste en MRI. La magnetoferritina de la invención es biocompatible y presenta una biodistribución en distintos órganos, mejorando significativamente las propiedades de otros agentes de contraste como son la magnetita sola. Esta puede ser usada como agente de contraste bimodal en MRI. The inner cavity of the ferritin can be used to introduce magnetite, obtaining a superparamagnetic nanoparticle called magnetoferritin, from now on the magnetoferritin of the invention. This superparamagnetic nanoparticle is constituted by a magnetite nanoparticle encapsulated in ferritin that has properties to behave as a contrast agent in MRI. The magnetoferritin of the invention is biocompatible and has a biodistribution in different organs, significantly improving the properties of other contrast agents such as magnetite alone. This can be used as a bimodal contrast agent in MRI.
Otras ventajas de la magnetoferritina de la presente invención es que la preparación de estas nanopartículas se puede ocluir 99mTc. Preferiblemente este se introduce en forma de 99mTcO4 ". La introducción de 99mTcO4 se puede llevar a cabo a temperatura ambiente en un tiempo óptimo para su inyección en el cuerpo. Además no requiere reducción del Tc(VII) y su incorporación a la nanopartícula es prácticamente total, lo que permite una acumulación óptima del radionúclido. El hecho de que el pertecnectato vaya ocluido en la red del mineral de Fe, hace que pueda controlarse la cantidad de 99mTcO4 " por partícula y por lo tanto, puedan prepararse fármacos de diferentes dosis de radionúclido, en función de las necesidades. Al introducirse el 99mTc hace que las magnetoferritina de la invención se pueden usar como agente de contraste trimodal, OI-MRI-SPECT. Las especies de Te preferidas para su inclusión en la magnetoferritina de la invención es 99mTcO4 ". La concentración de 99mTc se puede ajustar en función de la necesidad, por ejemplo diagnosis o terapia pero preferiblemente está comprendida entre 10"9-10"5M. Los presentes inventores han logrado obtener partículas de magnetita de alto contenido en Fe en la cavidad de ferritina. El contenido en Fe puede ser modulado hasta 3800 átomos de Fe por unidad de ferritina. Preferiblemente se modula la cantidad de Fe de 3000 a 3800 átomos de Fe por unidad de ferritina, lo cual permite mejora sus propiedades como agente de contraste en MRI. Other advantages of the magnetoferritin of the present invention is that the preparation of these nanoparticles can be occluded 99m Tc. Preferably this is introduced in the form of 99m TcO 4 " . The introduction of 99m TcO 4 can be carried out at room temperature at an optimal time for injection into the body. In addition it does not require reduction of Tc (VII) and its incorporation into the nanoparticle is practically total, which allows an optimal accumulation of the radionuclide. The fact that the pertecnectate is occluded in the Fe ore network makes it possible to control the amount of 99m TcO 4 " per particle and therefore, can Preparing drugs of different doses of radionuclide, depending on the needs. At the introduction of 99m Tc, the magnetoferritins of the invention can be used as a trimodal contrast agent, OI-MRI-SPECT. The preferred Te species for inclusion in the magnetoferritin of the invention is 99m TcO 4 " . The concentration of 99m Tc can be adjusted according to need, for example diagnosis or therapy but preferably is between 10 " 9 -10 " 5 M. The present inventors have managed to obtain magnetite particles of high Fe content in the ferritin cavity.The Fe content can be modulated up to 3800 Fe atoms per unit of ferritin Preferably, the amount of Fe from 3000 to 3800 Fe atoms per unit of ferritin, which improves its properties as a contrast agent in MRI.
Durante la preparación de la nanopartícula de magnetita (i), el Te queda ocluido dentro de la magnetita, lo que es un ventaja frente a los complejos de coordinación de Te puesto que la concentración de Te por partícula es mucho mayor (hasta 20 veces mayor) y permite acumular una mayor densidad de átomos de Te. Esta mayor acumulación redunda en un incremento de la radiación gamma y en una mayor resolución. Asimismo, la acumulación optimiza la concentración del radiomarcador, lo que permite el uso de menores dosis. During the preparation of the magnetite nanoparticle (i), the Te is occluded within the magnetite, which is an advantage over the complexes of coordination of Te since the concentration of Te per particle is much higher (up to 20 times higher) and allows a greater density of Te atoms to accumulate. This greater accumulation results in an increase in gamma radiation and a higher resolution. Also, the accumulation optimizes the concentration of the radiolabel, which allows the use of lower doses.
Los datos experimentales han demostrado que después de 3 h de inyección, las partículas se acumulan de forma significativa en pulmón (Figura 2). Por otra parte, puesto qué controlar la cantidad de Tc/ferritina (en un rango 0-20, con un 100% prácticamente de incorporación de Te) permite que se pueda alcanzar de sobra el rango óptimo de concentraciones de Te (10"9 M) y el de MRI (del orden de 10"5 M). Otro de los aspectos es proporcionar un método para la síntesis de las ferritinas y las magnetoferritinas de la presente invención. Este método comprende: a) anclar al menos un quantum dot a la superficie de la ferritina b) y después anclar las cadenas de PEG a la superficie de la ferritina. Experimental data has shown that after 3 h of injection, the particles accumulate significantly in the lung (Figure 2). On the other hand, since controlling the amount of Tc / ferritin (in a range 0-20, with 100% practically incorporating Te) allows the optimum range of concentrations of Te (10 "9 M) to be exceeded. ) and that of MRI (of the order of 10 "5 M). Another aspect is to provide a method for the synthesis of ferritins and magnetoferritins of the present invention. This method comprises: a) anchoring at least one quantum dot to the surface of the ferritin b) and then anchoring the PEG chains to the surface of the ferritin.
En una realización preferida la etapa (a) comprende: adicionar una disolución que comprende un quantum dot derivatizado con grupos carboxílicos en la superficie y un agente de acoplamiento, como una carboimida; a otra disolución de la ferritina y posteriormente hacer reaccionar la disolución que comprende el PEG. In a preferred embodiment step (a) comprises: adding a solution comprising a quantum dot derivatized with carboxylic groups on the surface and a coupling agent, such as a carboimide; to another solution of the ferritin and subsequently reacting the solution comprising the PEG.
La introducción de las cadenas de polímero biocompatible se deben de realizar después de la introducción del QD. La introducción del polímero biocompatible se realiza preferentemente mediante el uso de derivados del polímero biocompatible que comprenden ácidos carboxílicos, y así se forman enlaces tipo amida, por lo que se puede usar un agente de acoplamiento para la catálisis de la reacción. El biopolímero puede estar activado/funcionalizado, es decir los ácidos carboxílicos pueden estar derivatizados, incluyendo ya el agente de acoplamiento. The introduction of biocompatible polymer chains must be carried out after the introduction of the QD. The introduction of the biocompatible polymer is preferably carried out by using biocompatible polymer derivatives comprising carboxylic acids, and thus bonds are formed. amide type, so a coupling agent can be used for catalysis of the reaction. The biopolymer may be activated / functionalized, that is, the carboxylic acids may be derivatized, including the coupling agent.
Los agentes de acoplamiento preferidos de la presente invención se selecciona entre N,N-diciclohexilcarbodiimida, 1 -[3-(dimetilamino)propil]-3-etilcarbodiimida, diisopropilcarbodiimida y cualquiera de sus combinaciones. Respecto la síntesis de la magnetoferritina, esta se realiza antes de la introducción del QD y del polímero biocompatible. Aunque la introducción de la magnetita se puede hacer mediante el proceso descrito por Douglas (Masaki Uchida, Masahiro Terashima, Charles H. Cunningham, Yoriyasu Suzuki, Deborah A. Willits, Ann F. Willis, Philip C. Yang, Philip S. Tsao, Michael V. McConnell, Mark J. Young and Trevor Douglas, Magnetic Resonance in Medicine 60:1073-1081 (2008)), los presentes inventores han desarrollado una variación, que proporcionan algunas ventajas. El método de la invención comprende al menos las etapas de: i) preparar una disolución de apoferritina, preferiblemente de concentración entre 0,1 y 100 mg/mL, y más preferiblemente entre 5 y 20 mg/mL, ii) Preparar una disolución que comprenda Fe(ll) y Fe(lll) en estequiometría aproximada de 1 :2, es decir entre 1 :1 ,8 a 1 ,8:1 iii) preparar una disolución de pertecnectato (99mTc) iv) adicionar de forma intercalada la disolución preparada en el paso (ii) y la disolución preparada en la etapa (iii) sobre la preparada en el paso (i). v) Preferiblemente, se aisla la magnetoferritina superparamagnética dopada con 99mTc La estequiometría inicial de Fe(ll) y Fe(lll) es determinante para la preparación apropiada de magnetoferritina y por ende de sus propiedades magnéticas. A diferencia del método reportado por Douglas y colaboradores, dónde todo el Fe de partida está en su estado de oxidación Fe(ll) y se ha comprobado que con un riguroso control de la extensión de la oxidación, en nuestro método, el balance estequiométrico de partida confiere las condiciones óptimas y no requiere un control exhaustivo de la entrada de aire en el sistema. Las sales de hierro (II) y (III) útiles para la preparación de la magnetoferritina son conocidas por los expertos en la materia. En una realización preferida la etapa (ii) se prepara mezclando una disolución que comprende sulfato de hierro (II) amoníaco hexahidratado con otra que comprende Fe (N03)3 en HCI. La inclusión de 99mTc04" se lleva a cabo mediante pequeñas adiciones de 99mTc04" al mismo tiempo que se hace la red de magnetita. Se puede dializar la disolución resultante en bolsas de diálisis con tamaño de poro adecuado y separar las partículas de todo el material restante. En la disolución que no contiene las partículas se puede medir la concentración de Te y de esta forma se conoce el porcentaje de Te incorporado. En algunos casos se ha estudiado el comportamiento del Mo04 2", porque tiene una química muy similar a del 99mTc04 ", pero no es radioactivo. Se ha observado que para pequeños contenidos de Mo (0-20 átomos de Mo/magnetoferritina), la incorporación es prácticamente del 100%. Preferred coupling agents of the present invention are selected from N, N-dicyclohexylcarbodiimide, 1 - [3- (dimethylamino) propyl] -3-ethylcarbodiimide, diisopropylcarbodiimide and any combination thereof. Regarding the synthesis of magnetoferritin, this is done before the introduction of the QD and the biocompatible polymer. Although the introduction of magnetite can be done through the process described by Douglas (Masaki Uchida, Masahiro Terashima, Charles H. Cunningham, Yoriyasu Suzuki, Deborah A. Willits, Ann F. Willis, Philip C. Yang, Philip S. Tsao, Michael V. McConnell, Mark J. Young and Trevor Douglas, Magnetic Resonance in Medicine 60: 1073-1081 (2008)), the present inventors have developed a variation, which provide some advantages. The method of the invention comprises at least the steps of: i) preparing an apoferritin solution, preferably of a concentration between 0.1 and 100 mg / mL, and more preferably between 5 and 20 mg / mL, ii) Preparing a solution that comprise Fe (ll) and Fe (lll) in stoichiometry of approximately 1: 2, that is, between 1: 1, 8 to 1, 8: 1 iii) prepare a solution of pertecnectate ( 99m Tc) iv) add the interleaved solution prepared in step (ii) and the solution prepared in step (iii) over that prepared in step (i). v) Preferably, the superparamagnetic magnetoferritin doped with 99m Tc is isolated The initial stoichiometry of Fe (ll) and Fe (lll) is decisive for the proper preparation of magnetoferritin and hence its magnetic properties. Unlike the method reported by Douglas et al., Where all the initial Fe is in its oxidation state Fe (ll) and it has been proven that with a strict control of the extent of oxidation, in our method, the stoichiometric balance of departure confers optimal conditions and does not require exhaustive control of the air inlet in the system. The iron (II) and (III) salts useful for the preparation of magnetoferritin are known to those skilled in the art. In a preferred embodiment step (ii) is prepared by mixing a solution comprising iron (II) sulfate ammonia hexahydrate with another comprising Fe (N0 3 ) 3 in HCI. The inclusion of 99m Tc0 4 " is carried out by small additions of 99m Tc0 4" at the same time as the magnetite network is made. The resulting solution can be dialyzed into dialysis bags with adequate pore size and separate particles from all remaining material. In the solution that does not contain the particles, the concentration of Te can be measured and in this way the percentage of Te incorporated is known. In some cases the behavior of Mo0 4 2 " has been studied, because it has a chemistry very similar to 99m Tc0 4 " , but it is not radioactive. It has been observed that for small Mo contents (0-20 Mo / magnetoferritin atoms), the incorporation is practically 100%.
Con objeto de llevar una caracterización más pormenorizada que permita conocer la distribución de Te en la nanopartícula superparamagnética, se llevó a cabo un estudio mediante Microscopía Electrónica de Transmisión de muestras similares en las que en vez de usar el radiomarcador pertecnectato, se usaron otros aniones del tipo molibdato, vanadato, arseniato y fosfato. La química de estos aniones es similar a la del pertecnectato, especialmente si no hay cambios del estado de oxidación del metal. El estudio de Microscopía Electrónica de Transmisión es de especial utilidad en el caso de las nanopartículas dopadas con vanadato; esta técnica nos proporciona tanto información sobre la distribución del tamaño de las nanopartículas como sobre la composición química y distribución espacial de los estados de oxidación en nanopartículas individuales. Las imágenes de campo oscuro a alto ángulo son sensibles al número atómico del material, es decir, aquellas zonas en la que estén presentes los elementos más pesados se mostrarán en la imagen como puntos de mayor intensidad. En nuestro caso de estudio, las nanopartículas, al contener elementos de alto número atómico, aparecerán en la imagen con alto contraste, siendo visualizadas directamente y de forma individual. En estas condiciones de registro de la imagen se puede realizar medidas directas del tamaño de las nanopartículas presentes en la muestra, a partir de las cuales se puede establecer una distribución de tamaños de partícula. Adicionalmente se puede determinar la distribución espacial, con resolución sub-nanométrica, de los elementos químicos presentes en las partículas individuales, utilizando la técnica de pérdida de energía de los electrones (EELS). Esta técnica nos permite estudiar directamente las transiciones electrónicas que ocurren en el átomo cuando este es sometido a un haz de electrones de alta energía, 200kV. En particular esta técnica mide la energía que el electrón incidente pierde cuando interacciona con un átomo. Por ejemplo en el caso de los átomos de Vanadio, se estudian directamente las transiciones L2,3 donde los electrones 2p del átomo son transferidos a estados no ocupados sobre el nivel de Fermi. La energía requerida para esta transición es un valor característico para cada átomo y es igual a la energía pérdida por el electrón incidente. Así, midiendo la pérdida de energía de los electrones incidentes uno puede identificar los distintos elementos presentes en las nanopartículas, (V 513eV, O 532eV, Fe 708eV). Adicionalmente la estructura fina del espectro EELS de los metales de transición se caracteriza por la presencia de dos picos intensos (líneas blancas) cuya intensidad y posición en energía varía en función del estado de oxidación del material (Leapman et al Phys. Rev. Lett. 45, 397 (1980), Turquat et al. International Journal of Inorganic Materials 3 (2001 ) 1025-1032). El estudio detallado de la estructura fina del pico de absorción refleja información sobre el estado electrónico del material pudiendo estudiar las posibles variaciones del estado de oxidación del hierro o vanadio a través de las nanopartículas. In order to carry out a more detailed characterization that allows to know the distribution of Te in the superparamagnetic nanoparticle, a study was carried out by means of Transmission Electron Microscopy of similar samples in which instead of using the pertechnectate radiolabel, other anions of the type molybdate, vanadate, arsenate and phosphate. The chemistry of these anions is similar to that of pertecnectate, especially if there are no changes in the oxidation state of the metal. The study of Transmission Electron Microscopy is especially useful in the case of nanoparticles doped with vanadate; This technique provides us with information about the nanoparticle size distribution as well as the chemical composition and spatial distribution of oxidation states in individual nanoparticles. Dark-field images at a high angle are sensitive to the atomic number of the material, that is, those areas where the heaviest elements are present will be shown in the image as points of greater intensity. In our case study, the nanoparticles, containing elements of high atomic number, will appear in the image with high contrast, being displayed directly and individually. Under these conditions of image registration, direct measurements of the size of the nanoparticles present in the sample can be made, from which a particle size distribution can be established. Additionally, the spatial distribution can be determined, with sub-nanometric resolution, of the chemical elements present in the individual particles, using the electron energy loss technique (EELS). This technique allows us to directly study the electronic transitions that occur in the atom when it is subjected to a beam of high-energy electrons, 200kV. In particular, this technique measures the energy that the incident electron loses when it interacts with an atom. For example in the case of Vanadium atoms, the L2.3 transitions are directly studied where the 2p electrons of the atom are transferred to unoccupied states above the Fermi level. The energy required for this transition is a characteristic value for each atom and is equal to the energy lost by the incident electron. Thus, by measuring the energy loss of the incident electrons one can identify the different elements present in the nanoparticles, (V 513eV, O 532eV, Fe 708eV). Additionally, the fine structure of the EELS spectrum of transition metals is characterized by the presence of two intense peaks (white lines) whose intensity and position in energy varies depending on the oxidation state of the material (Leapman et al Phys. Rev. Lett. 45, 397 (1980), Turquat et al. International Journal of Inorganic Materials 3 (2001) 1025-1032). The detailed study of the fine structure of the absorption peak reflects information on the electronic state of the material and can study the possible variations in the oxidation state of iron or vanadium through the nanoparticles.
El estudio de la composición química y los estados de oxidación en nanopartículas individuales fue llevado a cabo combinando las propiedades de las imágenes en campo oscuro a alto ángulo con la espectroscopia de pérdida de energía de los electrones, utilizando el método de adquisición conocido como esprectro-imagen (Tence, M. Quartuccio and C. Colliex, Ultramicroscopy 58 (1995) 42, Maigne et al Journal of Electron Microscopy 58(3): 99-109 (2009)). Este modo consiste en adquirir simultáneamente la señal de campo oscuro a alto ángulo y los espectros EELS mientras la sonda barre una zona predeterminada, imagen (1 D) o espectro línea. En particular utilizando un tiempo de adquisición de 2 segundos se adquirió un espectro EELS, con energía de dispersión de 0,5eV, cada 0,6 nm a lo largo de una línea de 36,7nm que pasa a través de las nanopartículas. El análisis de cada uno de los espectros adquiridos (cuantificación y estudio del estado de oxidación) a lo largo de la nanopartícula nos proporciona la composición y estado de oxidación del metal caracterizado a la escala subnanométrica. The study of the chemical composition and oxidation states in individual nanoparticles was carried out by combining the properties of the dark field images at high angle with the electron energy loss spectroscopy, using the acquisition method known as esprectro- image (Tence, M. Quartuccio and C. Colliex, Ultramicroscopy 58 (1995) 42, Maigne et al Journal of Electron Microscopy 58 (3): 99-109 (2009)). This mode consists of simultaneously acquiring the dark-field signal at a high angle and the EELS spectra while the probe sweeps a predetermined area, image (1 D) or line spectrum. In particular, using an acquisition time of 2 seconds, an EELS spectrum was acquired, with dispersion energy of 0.5eV, every 0.6 nm along a 36.7nm line that passes through the nanoparticles. The analysis of each of the acquired spectra (quantification and study of the oxidation state) along the nanoparticle gives us the composition and oxidation state of the metal characterized to the sub-nanometer scale.
Preferiblemente en el método en la disolución de la etapa (a) esta tamponada con AMPSO a un pH entre pH 7,5 y 9,5, preferiblemente entre 8,0 y 9,0. En una realización particular, las cadenas del polímero biocompatible, preferiblemente PEG, se hacen reaccionar con la magnetoferritina después de la inclusión de la magnetita/Tc. Preferably in the method in the solution of step (a) it is buffered with AMPSO at a pH between pH 7.5 and 9.5, preferably between 8.0 and 9.0. In a particular embodiment, the chains of the biocompatible polymer, preferably PEG, are reacted with the magnetoferritin after the inclusion of the magnetite / Tc.
Otro aspecto se refiere a una composición farmacéutica que comprende las magnetoferritina de la presente invención y al menos un excipiente farmacéuticamente aceptable, así como el uso de dicha composición farmacéutica para la preparación de un medicamento. Las ferritina de la presente invención, y especialmente la magnetoferritina, así como la composición farmacéutica que las incluye son útiles para la preparación de medicamento para la diagnosis de diferentes enfermedades, según la utilización de moléculas que lo confieran especificidad por un tejido u órgano en cuestión, pero en especial cáncer, incluyendo cáncer cervical, de cabeza y cuello, renal y de uréter, de colon, recto y ano, de endometrio, de esófago, de estómago, de hígado, de laringe, de ovario, de páncreas, de piel, de próstata, de pulmón, de cerebro, de testículo, leucemia, melanoma, y linfoma. Another aspect relates to a pharmaceutical composition comprising the magnetoferritins of the present invention and at least one pharmaceutically acceptable excipient, as well as the use of said pharmaceutical composition for the preparation of a medicament. The ferritin of the present invention, and especially the magnetoferritin, as well as the pharmaceutical composition that includes them, are useful for the preparation of medicament for the diagnosis of different diseases, according to the use of molecules that confer specificity for a tissue or organ in question. , but especially cancer, including cervical, head and neck, renal and ureter, colon, rectum and anus, endometrial, esophagus, stomach, liver, larynx, ovarian, pancreas, skin cancer , prostate, lung, brain, testis, leukemia, melanoma, and lymphoma.
Además de para detectar enfermedades también son útiles tanto la ferritina de la presente invención, y especialmente magnetoferritina, así como la composición farmacéutica que las comprende como agentes de contraste en general y como agente de contraste en MRI, OI o SPECT. In addition to detecting diseases, both the ferritin of the present invention, and especially magnetoferritin, as well as the pharmaceutical composition comprising them as contrast agents in general and as a contrast agent in MRI, OI or SPECT are also useful.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Los siguientes ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. DESCRIPCIÓN DE LAS FIGURAS Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples are provided by way of illustration, and are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES
La figura 1 muestra una de las realizaciones particulares de la nanoestructura de la invención. Esta consta de ferritina (i), con un núcleo de magnetita dopado con 99mTc (iv), un quantum dot anclado a la superficie (ii), y polímero biocompatible de PEG (iii). La nanoestructura es capaz de actuar como agente de contraste en MRI (i), OI (ii), gammagrafía (iv) y muestra tiempos de vida medio en sangre suficientemente extensos para distribuirse por el sistema circulatorio sin ser fagocitados en un tiempo menor de 3h. Figure 1 shows one of the particular embodiments of the nanostructure of the invention. This consists of ferritin (i), with a magnetite core doped with 99m Tc (iv), a quantum dot anchored to the surface (ii), and biocompatible PEG polymer (iii). The nanostructure is capable of acting as a contrast agent in MRI (i), OI (ii), scintigraphy (iv) and shows life times medium in blood sufficiently extensive to be distributed through the circulatory system without being phagocyted in a time less than 3h.
La figura 2. TEM-STEM de la nanoestructura mostrando la formación preferencial de dímeros ferritina-QD. Figure 2. TEM-STEM of the nanostructure showing the preferential formation of ferritin-QD dimers.
La figura 3. EELS en línea de un dímero ferritina-QD, demostrando la presencia de Fe y O en el bloque de ferritina y de Cd en el de QD. La figura 4. Imagen de ratancillos atímicos a los que se le inyectó la nanoestructura objeto de la presente patente. Tras 3 h, se observa la fluorescencia de la nanoestructura, centrada preferencialmente en pulmones, con una concentración prácticamente despreciable en el hígado. Ejemplos. Figure 3. EELS in line of a ferritin-QD dimer, demonstrating the presence of Fe and O in the ferritin block and Cd in that of QD. Figure 4. Image of athletic ratancillos to which the nanostructure object of the present patent was injected. After 3 h, the fluorescence of the nanostructure, preferentially centered in the lungs, is observed, with a practically negligible concentration in the liver. Examples
Acoplamiento ferritina con Quantum Dots:: Ferritin coupling with Quantum Dots ::
Como ejemplo de una de nuestras realizaciones particulares, la ferritina se acopló con 3 tipos diferentes de quantum dots: un primer tipo de quantum dots emitiendo en el verde (QD525), un segundo tipo emitiendo en el rojo (QD655) y un tercero que emite en el cercano infrarrojo (QD800). Para ello se llevó a cabo una incubación directa de ambos tipos de nanopartículas (magnetoferritina y Qdot) en presencia del catalizador EDC (1 -etil-3-[3- dimetilaminopropil] carbodiimida), y se obtuvo una nanoestructura que contenía ambos tipos de nanoprecursores (magnetoferritina+Qdot). As an example of one of our particular embodiments, ferritin was coupled with 3 different types of quantum dots: a first type of quantum dots emitting in the green (QD525), a second type emitting in the red (QD655) and a third emitting in the near infrared (QD800). For this, a direct incubation of both types of nanoparticles (magnetoferritin and Qdot) was carried out in the presence of the EDC catalyst (1-ethyl-3- [3- dimethylaminopropyl] carbodiimide), and a nanostructure was obtained containing both types of nanoprecursors (magnetoferritin + Qdot).
El estudio por Optical Imaging de la biodistribución de la magnetoferritina acoplada al QD800 mostró lo siguiente: en un tiempo de entre 15 minutos y 1 hora después de la administración intravenosa la nanoestructura se concentra en hígado, pulmón y cerebro, en orden decreciente. Después de 3 horas, se localiza preferentemente en pulmones, siendo la concentración en hígado muy poco significativa (Figura 4). Tras 24 horas no se detectaron nanopartículas. The study by Optical Imaging of the biodistribution of magnetoferritin coupled to the QD800 showed the following: in a time between 15 minutes and 1 hour after intravenous administration the nanostructure is concentrated in the liver, lung and brain, in decreasing order. After 3 hours, it preferably located in the lungs, the concentration in the liver being very insignificant (Figure 4). After 24 hours no nanoparticles were detected.
Síntesis de maqnetoferritina-99mTc: Synthesis of maqnetoferritin- 99m Tc:
Disolución 1 . Se preparan 10 mi una disolución de apoferritina (Sigma-Aldrich Ref. A341 -1 G, lot. 048K7004) de concentración 10mg/ml en tampón AMPSO pH 8,6 (Sigma A6659). La disolución se desgasifica con una corriente fuerte de argón y en agitación durante 10 min. Dissolution 1. A solution of apoferritin (Sigma-Aldrich Ref. A341 -1 G, lot. 048K7004) with a concentration of 10mg / ml in AMPSO buffer pH 8.6 (Sigma A6659) is prepared 10 ml. The solution is degassed with a strong stream of argon and under stirring for 10 min.
Disolución 2. Se preparan dos disoluciones: 5 mi de Sal de Mohr (Amonium Iron (II) sulfate hexahydrate, Aldrich Chem. 20,350-5) 0,05 M en HCI 0,01 M y 5 mi de Fe (N03)3 0,1 M en HCI 0,01 M, se mezclan y se desgasifica con una corriente fuerte de argón y en agitación durante 10 min. Solution 2. Two solutions are prepared: 5 ml of Mohr Salt (Amonium Iron (II) sulfate hexahydrate, Aldrich Chem. 20,350-5) 0.05 M in 0.01 M HCI and 5 ml of Fe (N0 3 ) 3 0.1 M in 0.01 M HCI, mixed and degassed with a strong argon stream and stirred for 10 min.
Disolución 3. Una disolución de NaOH 0,1 M se desgasifica con una corriente fuerte de argón y en agitación durante 10 min. Solution 3. A solution of 0.1 M NaOH is degassed with a strong stream of argon and under stirring for 10 min.
Disolución 4. Una disolución de pertenectato (99mTc) obtenida a partir de kit comercial se desgasifica con una corriente fuerte de argón y en agitación durante 2 min. Solution 4. A solution of a particulate ( 99m Tc) obtained from a commercial kit is degassed with a strong stream of argon and under stirring for 2 min.
Suspensión 5. Se lleva a cabo la adición lenta de la disolución 2 sobre la disolución 1 . Se llevan a cabo adiciones de 0,25 mi cada 2 min hasta completar 1 mi. Antes de la 5 adición de la disolución 2, se adicionan 56 μΙ de la disolución de 99mTc (disolución 4). Suspension 5. Slow addition of solution 2 over solution 1 is carried out. Additions of 0.25 ml are made every 2 min until 1 ml is completed. Before the addition of solution 2, 56 μΙ of the 99m Tc solution (solution 4) is added.
Disolución 6. A la suspensión 5 se le adiciona lentamente 1 mi de una disolución de citrato sódico 0,1 M para eliminar todo los compuestos metálicos que no hayan quedado encapsulados en la apoferritina. La disolución resultante se cromatografía (10 min) en columna de exclusión por tamaño (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17- 0851 -01 ), obteniendo la disolución final 6 que contiene mangnetita dopada con 99mTc encapsulada en la cavidad de la apoferritina. Solution 6. Suspension 5 is slowly added 1 ml of a 0.1 M sodium citrate solution to remove all metal compounds that have not been encapsulated in apoferritin. The resulting solution is chromatographed (10 min) in size exclusion column (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17- 0851 -01), obtaining the final solution 6 containing mangnetite doped with 99m Tc encapsulated in the apoferritin cavity.
Adición del radioisótopo de Te a la maqnetoferritina. Addition of the radioisotope of Te to maqnetoferritin.
Disolución 4. Se usa un generador de 99Mo/99mTc de 12 GBq de actividad calibrada. La elución realizada es analizada en términos de actividad de 99mTc. Conocida la relación específica mCi^g de 99mTc, es posible controlar la cantidad de Te utilizada y por ende su actividad radioquímica. Solution 4. A 99 Mo / 99m Tc generator with 12 GBq of calibrated activity is used. The elution performed is analyzed in terms of 99m Tc activity. Once the specific ratio mCi ^ g of 99m Tc is known, it is possible to control the amount of Te used and therefore its radiochemical activity.
Suspensión 5. Se lleva a cabo la adición lenta de la disolución 2 sobre la disolución 1 . Se llevan a cabo adiciones de 0,25 mi cada 2 min hasta completar 1 mi. Antes de la 5 adición de la disolución 2, se adicionan 0,5 mi de la disolución de 99mTc (disolución 4). Suspension 5. Slow addition of solution 2 over solution 1 is carried out. Additions of 0.25 ml are made every 2 min until 1 ml is completed. Before the addition of solution 2, 0.5 ml of the 99m Tc solution (solution 4) is added.
Para la realización del control de calidad usamos tiras de papel Whatman 3 MM de 10 cm de longitud y 0,5 cm de ancho, en las que depositamos una alícuota (150 μΙ) del radiofármaco marcado, y para su desarrollo las introducimos en tanques cromatográficos con acetona hasta unos 0,5 mm de la base. Cuando la cromatografía se ha desarrollado la medimos en el Radio Cromatógrafo Minigita Raytest. En el origen de la tira cromatográfica se quedarán los coloides (Rf=0), en este caso la magnetoferritina dopada con Te, y en el frente el pertecnetato libre (Rf=1 ) Acoplamiento de la maqnetoferritina-99mTc (disolución 6) con Quantum Dots: For quality control, we use Whatman 3 MM paper strips 10 cm long and 0.5 cm wide, in which we deposit an aliquot (150 μΙ) of the labeled radiopharmaceutical, and for its development we introduce them in chromatographic tanks with acetone up to about 0.5 mm from the base. When the chromatography has developed we measure it on the Raytest Minigit Chromatograph Radio. In the origin of the chromatographic strip, the colloids (Rf = 0) will remain, in this case the magnetoferritin doped with Te, and in the front the free pertechnetate (Rf = 1) Maqnetoferritin coupling - 99m Tc (solution 6) with Quantum Dots:
Los quantum dots QD525, QD655 y QD800 se adquirieron en Invitrogen (Q21341 MP, Q21321 MP, Q21371 MP, respectivamente). Los quantum dots están recubiertos por un polímero funcionalizado con grupos carboxilicos para reaccionar con los grupos amino de la ferritina. Los residuos carboxilicos de los QDs deben activarse con EDC (1 -etil-3-[3-dimetilaminopropil] carbodiimida, Fluka 03450-25G). Disolución 7. 20 μΙ de la solución stock del QD comercial (8 mM) se incuban con 10 μΙ de una disolución stock de EDC (10 mg/ml en agua bidestilada) durante 30 min para su activación. Esta preparación se lleva a cabo mientras se desgasifican las disoluciones requeridas en el paso anterior de la síntesis de magnetoferritina. Quantum dots QD525, QD655 and QD800 were purchased from Invitrogen (Q21341 MP, Q21321 MP, Q21371 MP, respectively). The quantum dots are coated by a polymer functionalized with carboxylic groups to react with the amino groups of the ferritin. The carboxylic residues of the QDs must be activated with EDC (1-ethyl-3- [3-dimethylaminopropyl] carbodiimide, Fluka 03450-25G). Solution 7. 20 μΙ of the commercial QD stock solution (8 mM) is incubated with 10 μΙ of a stock EDC solution (10 mg / ml in double distilled water) for 30 min for activation. This preparation is carried out while degassing the solutions required in the previous step of magnetoferritin synthesis.
Disolución 8. 0,2 mi de la disolución 6 (magnetoferritina-99mTc) se diluyen hasta 2 mi en tampón PBS (Fosfato potásico monobásico, Acros Organics 424205000 y fosfático sódico dibásico, Acros Organics 424375000) y se le añade la disolución 7 (quantum dot activado. La mezcla se incuba durante 1 h en agitación suave y a 4QC. Posteriormente la muestra se purifica en una columna de cromatografía (15 min) por exclusión de tamaño (Sephacryl 5,5 cm x 1 ,5 cm de lecho, Sigma) con el fin de eliminar el exceso de producto que no hubiese reaccionado. Solution 8. 0.2 ml of solution 6 (magnetoferritin- 99m Tc) is diluted to 2 ml in PBS buffer (monobasic potassium phosphate, Acros Organics 424205000 and dibasic sodium phosphate, Acros Organics 424375000) and solution 7 is added ( quantum dot activated The mixture is incubated for 1 h under gentle agitation at 4 Q C. Subsequently the sample is purified on a chromatography column (15 min) by size exclusion (Sephacryl 5.5 cm x 1.5 cm bed , Sigma) in order to eliminate excess product that had not reacted.
Acoplamiento de PEG PEG coupling
El derivado de polietilenglicol MeO-PEG-NHS a-Metoxi-Qj-carboxilico ácido succinimidil éster poli(etilenglicol) (PEG-MW 2.000 Dalton)/ M.W. 2000 g/mol) se adquirió en Iris Biotech GmbH (PEG1 1 64, lot. 125447). The polyethylene glycol derivative MeO-PEG-NHS a-Methoxy-Qj-carboxylic acid succinimidyl ester poly (ethylene glycol) (PEG-MW 2,000 Dalton) / M.W. 2000 g / mol) was purchased from Iris Biotech GmbH (PEG1 1 64, lot. 125447).
Disolución 9. 1000 moles de PEG (0.0058 g en 0.5 mi de agua bidestilada) fueron añadidos a la disolución 8 y se dejó 30 min en agitación suave y a temperatura ambiente. Se cromatografió (10 min) en una columna de exclusión por tamaño (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851 -01 ) hasta obtener una disolución pura de nanopartículas de magnetita dopadas con 99mTc, acopladas covalentemente con un QD y PEG. Solution 9. 1000 moles of PEG (0.0058 g in 0.5 ml of double distilled water) were added to solution 8 and left 30 min under gentle stirring and at room temperature. Chromatograph (10 min) on a size exclusion column (Sephadex G-25, lot.360710, GE Healthcare, PD-10 Desalting Columns, 17-0851 -01) until a pure solution of magnetite nanoparticles doped with 99m is obtained Tc, covalently coupled with a QD and PEG.
Cuantificación de los quantum dot anclados Solamente cuando la preparación se lleva a cabo en un fuerte exceso de QD, se logran observar nanopartículas del tipo QD-magnetoferritina-QD. El recuento del número de QD enlazados convalentemente a la magnetoferritina en la nanoestructura objeto de la presente invención se llevó a cabo mediante TEM (Microscopía Electrónica de Transmisión) (figura 2) y mediante Microscopía Electrónica de Barrido-Transmisión en modo Campo Oscuro Anular de Alto Ángulo (HAADF-STEM) (figura 2). En esta última técnica, la intensidad de la señal con la que se construye la imagen depende del número atómico (Z), de tal forma que en las zonas de la imagen en las que están presentes elementos químicos con mayor valor de Z, el brillo es mayor. La relación entre la intensidad en la imagen y el valor del número atómico es aproximadamente del tipo Z2 (S.J. Pennycook, D.E. Jesson, Ultramicroscopy, 1991 , 37, 14). Como puede observarse en la figura, la imagen STEM-HAADF muestra claramente la presencia de dos partículas de dimensiones nanométricas próximas entre sí. Puede apreciarse, igualmente, que hay una clara diferencia de brillo entre las dos partículas que forman la nanoestructura, tal como cabría esperar, puesto que una contiene un óxido de Fe (ZFe=26, Z0=8) y la otra, una capa de CdSe (ZCd= 48, Zse=34). De acuerdo con lo comentado anteriormente, la partículas que muestra menor intensidad sería la que contiene elementos más ligeros (Fe, O) y la más brillante aquella que contiene a los elementos más pesados (Cd, Se). Para confirmar la interpretación anterior y obtener una evidencia directa sobre la naturaleza química de cada una de las dos partículas que se integraban en las nanoestructuras detectadas mediante Microscopía Electrónica, se llevó a cabo un estudio complementario mediante la técnica denominada EELS (Espectroscopia de pérdida de energía de los electrones). Esta técnica, que se lleva a cabo en los microscopios electrónicos de transmisión, se registra la energía cinética que pierde el haz de electrones incidente al atravesar la muestra. Esta pérdida de energía corresponde a las interacciones inelásticas electrón-muestra. Parte de estas interacciones corresponden a excitaciones de electrones de los niveles internos de los átomos presentes en la muestra a niveles por encima del nivel de Fermi. Como las energías de estas transiciones son específicas de cada átomo, el registro del espectro de pérdida de energías constituye una huella dactilar de los elementos presentes en la muestra, permitiendo en definitiva realizar un análisis químico cualitativo y cuantitativo de la zona iluminada por el haz de electrones. Como el diámetro de las sondas de electrones que se pueden obtener en microscopios de tranmisión como el empleado para obtener la figura, dotado con cañón de emisión de campo o FEG, puede ser del order de tan solo unos pocos Angstroms, esta técnica permite realizar lo que viene a llamarse como nanoanálisis, es decir un análisis químico cualitativo y cuantitativo con una resolución espacial mejor que un manómetro. Es más, si formamos una de estas sondas de dimensiones nanométricas, controlamos su movimiento sobre la muestra y en paralelo realizamos un registro simultáneo de la señal STEM-HAADF y de los espectros EELS podemos trabajar en un modo denominado de espectro-imagen (en el caso en el que la muestra se barra a lo largo de dos direcciones, sobre un área) o de espectro-línea (cuando la sonda de electrones se barre siguiendo una trayectoria lineal desde un punto a otro de la muestra). Trabajando en estos modos obtenemos información no sólo de qué elementos están presentes y en qué cantidad sino, además, sobre la distribución espacial de los elementos presentes en la muestra, con resolución subnanométrica. Esta aproximación experimental de espectro-línea es la que se ha aplicado al estudio de la composición química de las nanoestructuras reivindicadas. En nuestro caso se ha realizado análisis EELS a lo largo de trayectorias lineales que, partiendo del extremo de una de las partículas y cruzándola, llegaban y cruzaban la segunda de las partículas, adquiriendo un espectro, con una sonda menor que 5Á, tras cada 5Á. Como puede observarse (Figura 3), los espectros correspondientes a la zona de la imagen correspondiente a la partícula más brillante muestran señales correspondientes al Cd, mientras que los espectros registrados en la zona de la imagen correspondiente a la partícula menos brillante, contienen señales de Fe y O, confirmando que la nanoestructura consiste en dímeros de magnetoferritina y QD. Quantification of anchored quantum dot Only when the preparation is carried out in a strong excess of QD, can nanoparticles of the QD-magnetoferritin-QD type be observed. The count of the number of QD convalently linked to the magnetoferritin in the nanostructure object of the present invention was carried out by TEM (Transmission Electron Microscopy) (Figure 2) and by Scanning-Transmission Electron Microscopy in High-Angle Dark Field Mode Angle (HAADF-STEM) (figure 2). In this last technique, the intensity of the signal with which the image is constructed depends on the atomic number (Z), so that in the areas of the image in which chemical elements with greater value of Z are present, the brightness is older. The relationship between the intensity in the image and the value of the atomic number is approximately of the Z 2 type (SJ Pennycook, DE Jesson, Ultramicroscopy, 1991, 37, 14). As can be seen in the figure, the STEM-HAADF image clearly shows the presence of two particles of nanometric dimensions close to each other. It can also be seen that there is a clear difference in brightness between the two particles that form the nanostructure, as one would expect, since one contains an oxide of Fe (Z F e = 26, Z 0 = 8) and the other, a layer of CdSe (Z C d = 48, Zse = 34). According to what has been commented above, the particles that show the lowest intensity would be the one that contains lighter elements (Fe, O) and the brightest is the one that contains the heaviest elements (Cd, Se). To confirm the previous interpretation and obtain direct evidence on the chemical nature of each of the two particles that were integrated into the nanostructures detected by Electron Microscopy, a complementary study was carried out using the technique called EELS (Energy loss spectroscopy of electrons). This technique, which is carried out in the transmission electron microscopes, records the kinetic energy that the incident electron beam loses as it passes through the sample. This loss of energy corresponds to the electron-sample inelastic interactions. Part of these interactions correspond to electron excitations of the internal levels of the atoms present in the sample at levels above the Fermi level. Since the energies of these transitions are specific to each atom, the record of the energy loss spectrum constitutes a fingerprint of the elements present in the sample, ultimately allowing a qualitative and quantitative chemical analysis of the area illuminated by the electron beam. As the diameter of the electron probes that can be obtained in transmission microscopes such as the one used to obtain the figure, equipped with a field emission cannon or FEG, can be of the order of only a few Angstroms, this technique allows which comes to be called nanoanalysis, that is to say a qualitative and quantitative chemical analysis with a spatial resolution better than a manometer. Moreover, if we form one of these probes of nanometric dimensions, we control their movement on the sample and in parallel we perform a simultaneous recording of the STEM-HAADF signal and of the EELS spectra we can work in a so-called spectrum-image mode (in the case in which the sample is swept along two directions, over an area) or spectrum-line (when the electron probe is swept along a linear path from one point to another in the sample). Working in these modes we obtain information not only of what elements are present and in what quantity but also about the spatial distribution of the elements present in the sample, with sub-nanometer resolution. This experimental spectrum-line approach is the one that has been applied to the study of the chemical composition of the claimed nanostructures. In our case, EELS analysis has been carried out along linear paths that, starting from the end of one of the particles and crossing it, reached and crossed the second of the particles, acquiring a spectrum, with a probe smaller than 5Á, after every 5Á . As can be seen (Figure 3), the spectra corresponding to the area of the image corresponding to the brightest particle show signals corresponding to the Cd, while the spectra recorded in the area of the image corresponding to the less bright particle, contain signals of Fe and O, confirming that the nanostructure consists of magnetoferritin and QD dimers.

Claims

REIVINDICACIONES
1 . - Una ferritina, caracterizada porque comprende al menos un quantum dot y al menos una molécula de un polímero biocompatible ambos unidos covalentemente a la superficie de la ferritina. one . - A ferritin, characterized in that it comprises at least one quantum dot and at least one molecule of a biocompatible polymer both covalently bonded to the surface of the ferritin.
2. - La ferritina según la reivindicación anterior, caracterizada porque el quantum dot emite entre 750 y 850 nm. 2. - The ferritin according to the preceding claim, characterized in that the quantum dot emits between 750 and 850 nm.
3.- La ferritina cualquiera de las reivindicaciones anteriores, donde el quantum dot están recubiertos por un polímero funcionalizado con grupos carboxílicos. 3. The ferritin any of the preceding claims, wherein the quantum dot are coated by a polymer functionalized with carboxylic groups.
4.- La ferritina según cualquiera las dos reivindicaciones anteriores, caracterizada porque el quantum dot está unido covalentemente a la superficie de la ferritina mediante enlaces amido. 4. The ferritin according to any of the two preceding claims, characterized in that the quantum dot is covalently bonded to the surface of the ferritin by means of amido bonds.
5. - La ferritina según cualquiera de las reivindicaciones anteriores, caracterizada porque comprende entre 1 y 2 unidades de quantum dots anclados a la superficie de la ferritina. 5. - The ferritin according to any of the preceding claims, characterized in that it comprises between 1 and 2 quantum dots units anchored to the surface of the ferritin.
6. - La ferritina según cualquiera de las reivindicaciones anteriores, caracterizada porque el polímero biocompatible es polietilén glicol. 6. - Ferritin according to any of the preceding claims, characterized in that the biocompatible polymer is polyethylene glycol.
7. - La ferritina según la reivindicación anterior, caracterizada porque es obtenible por la unión de PEG1 163 MeO-PEG-COO-Su a-Metoxi-oo-ácido carboxilico succinimidil éster poli(etilenglicol) PEG-WM 2,000 Dalton a la superficie de la ferritina. 7. - The ferritin according to the preceding claim, characterized in that it is obtainable by the union of PEG1 163 MeO-PEG-COO-Su a-Methoxy-oo-carboxylic acid succinimidyl ester poly (ethylene glycol) PEG-WM 2,000 Dalton to the surface of Ferritin
8. - La ferritina según cualquiera de las dos reivindicaciones anteriores, caracterizada porque el polietilén glicol está unido mediante enlaces covalentes de tipo amida. 8. - Ferritin according to any of the two preceding claims, characterized in that the polyethylene glycol is linked by covalent bonds of the amide type.
9.- La magnetoferritina superparamagnética según cualquiera de las tres reivindicaciones anteriores, caracterizada porque tiene entre 3 y 10 cadenas de polietilén glicol unidas covalentemente a la superficie de la magnetoferritina. 9. The superparamagnetic magnetoferritin according to any of the three preceding claims, characterized in that it has between 3 and 10 chains of polyethylene glycol covalently bonded to the surface of the magnetoferritin.
10.- La ferritina según cualquiera de las reivindicaciones anteriores, caracteriza porque el núcleo tiene magnetita ocluyendo 99mTc. 10. The ferritin according to any of the preceding claims, characterized in that the core has magnetite occluding 99m Tc.
1 1 . - La ferritina según la reivindicación anterior, caracterizada porque la especie de Te es 99mTc04 ". eleven . - The ferritin according to the preceding claim, characterized in that the Te species is 99m Tc0 4 " .
12. - La ferritina según cualquiera de las dos reivindicaciones anteriores, caracterizada porque la concentración de la especie 99mTc está comprendida entre 10"9-10"5M. 12. - Ferritin according to any of the two preceding claims, characterized in that the concentration of the 99m Tc species is between 10 "9 -10 " 5 M.
13.- Un método para la síntesis de cualquiera de las ferritinas como se definen en las reivindicaciones anteriores que comprende: a) anclar al menos un quantum dot a la superficie de la ferritina b) y después anclar las cadenas de polietilén glicol a la superficie de la ferritina. 13. A method for the synthesis of any of the ferritins as defined in the preceding claims comprising: a) anchoring at least one quantum dot to the surface of the ferritin b) and then anchoring the polyethylene glycol chains to the surface of ferritin.
14. - El método según la reivindicación anterior, donde la etapa (a) comprende: adicionar una disolución que comprende un quantum dot derivatizado con grupos carboxilicos en la superficie y una carboimida; a otra disolución de la ferritina y posteriormente hacer reaccionar la disolución que comprende el polietilén glicol. 14. - The method according to the preceding claim, wherein step (a) comprises: adding a solution comprising a quantum dot derivatized with carboxylic groups on the surface and a carboimide; to another solution of the ferritin and subsequently reacting the solution comprising the polyethylene glycol.
15. - El método según cualquiera de las dos reivindicaciones anteriores, donde la etapa (b) comprende un agente de acoplamiento. 15. - The method according to any of the two preceding claims, wherein step (b) comprises a coupling agent.
1 6. - El método según cualquiera de las dos reivindicaciones anteriores, caracterizado porque el agente de acoplamiento se selecciona entre N,N- diciclohexilcarbodiimida, 1 -[3-(dimetilamino)propil]-3-etilcarbodiimida, diisopropilcarbodiimida y cualquiera de sus combinaciones. 1 6. - The method according to any of the two preceding claims, characterized in that the coupling agent is selected from N, N- dicyclohexylcarbodiimide, 1 - [3- (dimethylamino) propyl] -3-ethylcarbodiimide, diisopropylcarbodiimide and any combination thereof.
17. - El método según cualquiera de las reivindicaciones 13 a 16 para la síntesis de cualquiera de las ferritinas como se definen en las reivindicaciones 10 a 12 que comprende antes de la incorporación del quantum dot al menos las siguientes etapas: i) preparar una disolución de apoferritina, preferiblemente de concentración entre 0,1 y 100 mg/mL, y más preferiblemente entre 5 y 20 mg/mL, ii) preparar una disolución que comprenda Fe (II) y Fe(lll) en estequiometría aproximada de 1 :2. iii) preparar una disolución de pertenectato (99mTc) iv) adicionar de forma intercalada la disolución preparada en el paso (ii) y la disolución preparada en la etapa (iii) sobre la preparada en el paso (i). v) aislar la magnetoferritina superparamagnética dopada con 99mTc 17. - The method according to any of claims 13 to 16 for the synthesis of any of the ferritins as defined in claims 10 to 12 which comprises before incorporation of the quantum dot at least the following steps: i) preparing a solution of apoferritin, preferably of a concentration between 0.1 and 100 mg / mL, and more preferably between 5 and 20 mg / mL, ii) prepare a solution comprising Fe (II) and Fe (lll) in stoichiometry of approximately 1: 2 . iii) prepare a solution of pentactate ( 99m Tc) iv) add intercalated the solution prepared in step (ii) and the solution prepared in step (iii) over that prepared in step (i). v) isolate the superparamagnetic magnetoferritin doped with 99m Tc
18. - El método según la reivindicación anterior en la que la disolución de la etapa (ii) se prepara mezclando una disolución que comprende sulfato de hierro (II) amoníaco hexahidratado con otra que comprende Fe (N03)3 en HCI. 18. - The method according to the preceding claim wherein the solution of step (ii) is prepared by mixing a solution comprising iron (II) sulfate ammonia hexahydrate with another comprising Fe (N0 3 ) 3 in HCI.
19. - El método según cualquiera de las dos reivindicaciones anteriores, donde la disolución de la etapa (i) esta tamponada con AMPSO a un pH entre pH 7,5 y 9,5, preferiblemente entre 8,0 y 9,0. 19. - The method according to any of the two preceding claims, wherein the solution of step (i) is buffered with AMPSO at a pH between pH 7.5 and 9.5, preferably between 8.0 and 9.0.
20.- Una composición farmacéutica que comprende la ferritina como se en cualquiera de las reivindicaciones 1 a 12 y al menos un excipiente farmacéuticamente aceptable. 20. A pharmaceutical composition comprising ferritin as in any one of claims 1 to 12 and at least one pharmaceutically acceptable excipient.
21 . - Uso de la composición farmacéutica según la reivindicación anterior para la preparación de un medicamento. twenty-one . - Use of the pharmaceutical composition according to the preceding claim for the preparation of a medicament.
22. - El uso de la composición según la reivindicación anterior para la preparación de un medicamento para la diagnosis de cáncer. 22. - The use of the composition according to the preceding claim for the preparation of a medicament for the diagnosis of cancer.
23. - El uso de la ferritina según cualquiera de las reivindicaciones 1 a 12 o de la composición farmacéutica según la reivindicación 20 para la preparación de un medicamento como agente de contraste. 23. - The use of ferritin according to any one of claims 1 to 12 or of the pharmaceutical composition according to claim 20 for the preparation of a medicament as a contrast agent.
24. - El uso ferritina según cualquiera de las reivindicaciones 1 a 12 o de la composición farmacéutica según la reivindicación 20 para la preparación de un medicamento para su uso como agente de contraste en MRI, OI o SPECT. 24. - The use of ferritin according to any one of claims 1 to 12 or of the pharmaceutical composition according to claim 20 for the preparation of a medicament for use as a contrast agent in MRI, OI or SPECT.
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