WO2011070212A2 - Nanostructures mutlifonctionnelles utilisées comme agents de diagnostic trimodal irm-oi-spect - Google Patents

Nanostructures mutlifonctionnelles utilisées comme agents de diagnostic trimodal irm-oi-spect 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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WO2011070212A3 (fr
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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

L'invention concerne des nanostructures mutlifonctionnelles qui consistent en de la ferritine, caractérisée en ce qu'elle comprend au moins un point quantique et au moins une molécule d'un polymère biocompatible tous deux liés de manière covalente à la surface de la ferritine. Elle concerne également un procédé destiné à obtenir ces nanostructures, ainsi que leur utilisation comme médicament et, de préférence, dans le diagnostic du cancer ou comme agent de contraste.
PCT/ES2010/070816 2009-12-11 2010-12-10 Nanostructures mutlifonctionnelles utilisées comme agents de diagnostic trimodal irm-oi-spect WO2011070212A2 (fr)

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WO2019084537A1 (fr) * 2017-10-29 2019-05-02 Rourk Christopher J Circuits de porte de transport d'électrons et procédés de fabrication, de fonctionnement et d'utilisation
US10817780B2 (en) 2017-10-29 2020-10-27 Christopher J. Rourk Electron transport gate circuits and methods of manufacture, operation and use

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WO2005102396A2 (fr) * 2004-04-20 2005-11-03 Emory University Nanostructures de multimodalite, procedes de fabrication et d'utilisation associes
WO2008115854A2 (fr) * 2007-03-19 2008-09-25 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Nanoparticules multifonctions, compositions et procédés d'utilisation correspondants

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WO2008115854A2 (fr) * 2007-03-19 2008-09-25 Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services Nanoparticules multifonctions, compositions et procédés d'utilisation correspondants

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019084537A1 (fr) * 2017-10-29 2019-05-02 Rourk Christopher J Circuits de porte de transport d'électrons et procédés de fabrication, de fonctionnement et d'utilisation
US10817780B2 (en) 2017-10-29 2020-10-27 Christopher J. Rourk Electron transport gate circuits and methods of manufacture, operation and use

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