WO2017174437A1 - Ceria nanoparticles for use in the treatment of hepatocellular carcinoma - Google Patents

Ceria nanoparticles for use in the treatment of hepatocellular carcinoma Download PDF

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Publication number
WO2017174437A1
WO2017174437A1 PCT/EP2017/057570 EP2017057570W WO2017174437A1 WO 2017174437 A1 WO2017174437 A1 WO 2017174437A1 EP 2017057570 W EP2017057570 W EP 2017057570W WO 2017174437 A1 WO2017174437 A1 WO 2017174437A1
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cerium oxide
crystal
oxide nanoparticle
albumin
diameter
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PCT/EP2017/057570
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French (fr)
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Wladimiro JIMÉNEZ POVEDANO
Víctor FRANCO PUNTES
Guillermo FERNÁNDEZ VARO
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Hospital Clínic De Barcelona
Fundació Institut Català De Nanociència I Nanotecnologia
Institució Catalana De Recerca I Estudis Avançats
Universitat De Barcelona
Centro De Investigación Biomédica En Red
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Publication of WO2017174437A1 publication Critical patent/WO2017174437A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/244—Lanthanides; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0038—Radiosensitizing, i.e. administration of pharmaceutical agents that enhance the effect of radiotherapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6933—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5138—Organic macromolecular compounds; Dendrimers obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01F—COMPOUNDS OF THE METALS BERYLLIUM, MAGNESIUM, ALUMINIUM, CALCIUM, STRONTIUM, BARIUM, RADIUM, THORIUM, OR OF THE RARE-EARTH METALS
    • C01F17/00—Compounds of rare earth metals
    • C01F17/20—Compounds containing only rare earth metals as the metal element
    • C01F17/206—Compounds containing only rare earth metals as the metal element oxide or hydroxide being the only anion
    • C01F17/224—Oxides or hydroxides of lanthanides
    • C01F17/235—Cerium oxides or hydroxides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/60—Compounds characterised by their crystallite size

Definitions

  • the present invention relates to the field of medicine, in particular to cancer treatment. It also relates to nanoparticles and conjugates comprising them, as well as to pharmaceutically or veterinary compositions for the treatment of cancer.
  • HCC Hepatocellular carcinoma
  • Sorafenib a receptor tyrosine kinase inhibitor
  • Sorafenib is a small molecule that inhibits tumor-cell proliferation and tumor angiogenesis.
  • nanoparticles have been administered to an A375 xenograft model of nude mice in order to test them in a melanoma model.
  • the data are summarized in ANN et al., "Downregulation of Tumor Growth and Invasion by Redox-Active
  • CNPs redox-active cerium oxide nanoparticles
  • Cerium oxide nanoparticles have emerged thus in biomedical applications due to their superoxide dismutase (SOD) and catalase mimetic activity. CNPs act as catalysts with mixed valence that can exist in a reduced (+3) or oxidized (+4) state.
  • SOD superoxide dismutase
  • cerium oxide nanopartides are toxic. As an example, Cheng et al., in "Cerium oxide nanopartides induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signalling pathways",
  • CNPs or any other nanopartides with low diameters (fewer than 7 nm) have low residence time in the body, and poor solubility (See Hak Soo et al., "Renal clearance of quantum dots", Nature biotechnology - 2007, vol. no. 25(10), pp.: 1 165-1 170).
  • toxic reagents such as hexamethylenetetramine; HMT, making them hazardous for biomedical applications if they are not separated from the nanopartides before, which sometimes is difficult or imply expensive procedures.
  • Inventors propose using particular cerium oxide nanopartides in the treatment of HCC, which surprisingly allowed increasing the survival of treated animals in a remarkably way. Moreover, proliferation of cancer cells was highly reduced when the nanopartides were administered to mammals with induced HCC. Thus, the treatment avoided tumour growth.
  • a first aspect the invention relates to single-crystal cerium oxide nanopartides of formula (I) for use in the treatment of hepatocellular carcinoma, formula (I) defined by:
  • NP is a cerium oxide nanoparticle with a crystal diameter measured by transmission electron microscopy (TEM) from 3 to 24 nm;
  • TEM transmission electron microscopy
  • A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone (PVP), and combinations thereof; and
  • n is an integer from 0 to 40
  • n is an integer from 1 to 12.
  • nanoparticles are conjugated with A, so that they are also called in the present invention as conjugates.
  • the invention provides the unexpected effect of conjugates with CNPs or of CNPs that can be safely applied to HCC treatment.
  • the conjugates or CNPs are effective due to a proper non- agglomeration (or non-aggregation) of the nanoparticles, and due to a size that, though relative small, can be retained in liver.
  • the invention relates to particular single-crystal cerium oxide nanoparticles of formula (I),
  • NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm;
  • A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof;
  • n is an integer from 1 to 12.
  • Yet another aspect of the invention is a conjugated single-crystal cerium oxide nanoparticle of formula (I):
  • NP is a cerium oxide nanoparticle with a crystal diameter from 8 to 24 nm;
  • A is a molecule of albumin
  • N is an integer from 5 to 40.
  • CNPs of Bushan et al. were synthesized by the hydrothermal method. By TEM images it was seen that the average size of CNPs was of 4.35 ⁇ 1 .07 nm. CNPs were then encapsulated inside albumin nanoparticles via a desolvation technique using ethanol as the desolvating agent and glutaraldehyde as a cross linking agent to obtain least aggregated spherical BCNPs with uniform distribution.
  • the single-crystal cerium oxide nanoparticles of the invention can be administered to mammals in several forms appropriated for administration.
  • another aspect of the invention is a pharmaceutical or veterinary composition comprising these nanoparticles of formula (I) as defined above, together with one or more pharmaceutically or veterinary acceptable excipients or carriers.
  • These pharmaceutical or veterinary compositions comprising the new single-crystal cerium oxide nanoparticles of the invention are also for use in the treatment of hepatocellular carcinoma.
  • FIG. 1 shows in (A) optical microscope images (200X) of Ki-67-tincted hepatic cells from Wistar rats treated with cerium oxide nanoparticles (CeO2NPs) or with vehicle, which was a suspension of TMAOH 0.8 mM in phosphate saline buffer (PBS).
  • FIG. 1 shows in (A) optical microscope images (200X) of Ki-67-tincted hepatic cells from Wistar rats treated with cerium oxide nanoparticles (CeO2NPs) or with vehicle, which was a suspension of TMAOH 0.8 mM in phosphate saline buffer (PBS).
  • PBS phosphate saline buffer
  • Example 2 is a graphic with the survival (percentage of survival; %S) of the rats versus the time (T, weeks (W)) after reception of last dose of single-crystal cerium oxide nanoparticles of formula (I) in which A is albumin (CeO2NPs) or of vehicle as disclosed for FIG. 1 .
  • FIG. 3 related with Example 1 depicts the monitoring of the synthesis kinetics of CeO2NPs (single-crystal cerium oxide nanoparticles of formula (I) in which A is either albumin or PVP).
  • A the pH evolution of the synthesis solution.
  • B UV-visible spectrum of initial cerium (III) precursor (black line); as-synthesized NPs at 1 h of reaction, diluted 1 /20 (soft grey line); as-synthesized NPs at 48h of reaction, diluted 1 /20 (dark grey line).
  • FIG. 4 shows the characterization of PVP- conjugated CeO2NPs (single-crystal cerium oxide nanoparticles of formula (I) in which A is PVP) by transmission electron microscopy (TEM).
  • TEM transmission electron microscopy
  • FIG. 4 a high resolution TEM micrograph (HR-TEM) at high (400.000X) magnification, revealing the atomic planes of single-crystal nanoparticles
  • FIG. 4 b High Angle Annular Dark Field-Scanning Transmission Electron Microscopy (STEM-HAADF) image
  • STEM-HAADF High Angle Annular Dark Field-Scanning Transmission Electron Microscopy
  • FIG. 4 c HR-TEM image at low magnification (7.000 check); in FIG.
  • FIG. 4 d atomic resolution HR-TEM image of one single (single-crystal) CeO2NP, showing spherical morphology and the correspondent atomic planes; in FIG. 4 e) Fast Fourier Transformation (FFT) digital diffractogram calculated from the particle shown in FIG. 4 d); in FIG. 4 f) Electron energy-loss spectroscopy (EELS, X-axis Energy in kiloelectrovolts (keV); Y-axis counts (C) in arbitrary units (a.u.)) map of the chemical composition of as synthesized CeO2NP. Panels d), e) and F appear in Cont.FIG. 4 as a magnified image of squared single-crystal nanoparticle of panel c) in FIG. 4.
  • FFT Fast Fourier Transformation
  • FIG. 5, related with Example 1 is an X-ray diffraction (XRD) pattern of a single-crystal CeO2 nanoparticle of formula (I) with 5 nm of crystal size diameter.
  • XRD X-ray diffraction
  • FIG. 6 depicts the Dynamic Light Scattering (DLS) of BSA-coated CeO2NPs of the invention (single-crystal cerium oxide nanoparticles of formula (I) in which A is albumin).
  • DLS Dynamic Light Scattering
  • FIG. 6 a DLS of the as- synthesized BSA-coated (1 mM) CeO 2 NPs, by Intensity.
  • FIG. 6 b DLS of the as-synthesized BSA-coated (1 mM) CeO 2 NPs, by Number.
  • FIG. 6 c DLS of the supernatant only, containing free BSA, by Intensity.
  • FIG. 6 is an image of X ray contrast imaging of samples at different CeO2NPs concentrations ranging from 0 to 100 mg/ml .
  • FIG. 8 is a graphic showing the CeO2NPs antioxidant effect on fluorescently- labeled hydrogen peroxidase (EuTc-H2O2 complex),
  • (F) is fluorescence and (t) time in minutes(min).
  • nanoconjugate refers to a single-crystal cerium oxide nanoparticle which is attached to another compound selected from polyvinylpyrrolidone and proteins, in particular globular proteins, such as albumin.
  • conjugate also referred to as NP-A, refers to a single-crystal cerium oxide nanoparticle (NP) which is adsorbed by coordinate bonding to a molecule (A), that can be PVP and/or albumin.
  • single-crystal cerium oxide nanoparticle it is to be understood that said nanoparticle is not aggregated (by weak physical interaction) with other nanoparticles, in such a way that the nanoconjugate comprises only one nanoparticle as a core of the conjugate and of a particular crystal size diameter (or simply crystal diameter). All these structures can be seen by X-Ray diffraction and TEM. Thus, single-crystal or monocrystal means that there is only one crystal of cerium oxide configuring the nanopartide, with a particular crystal diameter.
  • nanopartide refers to a particle with at least two dimensions at the nanoscale, particularly with all three dimensions at the nanoscale, where the nanoscale is the range about 1 nm to about 100 nm, more particularly from 1 to 50 nm, and even more particularly from 5 to 20 nm.
  • Preferred crystal diameters of the nanopartides of the invention are from 5 to 10 nm (including, 5, 6, 7, 8, 9, and 10 nm), more preferably 5 nm.
  • shape of the nanopartides described herein there are included spheres and polyhedral.
  • the nanopartide is spherical.
  • the “nanopartide” refers to a particle with at least two dimensions at the
  • nanoscale this two dimensions being the cross-section of the nanopartide.
  • the term "size" refers to a characteristic physical dimension.
  • the size of the nanopartide corresponds to the diameter of the nanopartide.
  • the set of nanopartides can have a distribution of sizes around the specified size.
  • nanopartides can refer to a mode of a distribution of sizes, such as a peak size of the distribution of sizes.
  • the diameter is the equivalent diameter of the spherical body including the object.
  • cerium oxide refers to cerium (III) oxide (Ce 3+ ) and cerium (IV) oxide (Ce 4+ ) species that are both present when constituting the nanopartides. Although many of the cerium oxide nanopartides are usually in the (Ce 4+ ) oxidation state, small cerium oxide nanopartides are also in the (Ce 3+ ) oxidation state. Cerium oxide nanopartides (abbreviated also as Ceria- NP, CeO2-NP or simply CNP) are used in a variety of applications mainly due to its high surface area and the ability of cerium oxide to cycle between (III and IV) oxidation states. In a particular embodiment, cerium oxide
  • nanopartides are in the (Ce 4+ ) oxidation state
  • the diameter of the nanoparticle relates to the crystal size diameter (single-crystal).
  • Crystal size is usually measured from X-ray diffraction patterns while particle size is measured by TEM. In the case of single-crystal nanoparticles, XRD and TEM sizes coincide.
  • Typical crystal size diameters of the nanoparticles used in the present invention range from 3 to 24 nm (including 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 nm), when measured by TEM, more particularly the crystal size diameter of the nanoparticles is about 5 nm.
  • hydrodynamic diameter determined by dynamic light scattering (DLS) techniques based on the Stoke-Einstein equation is used herewith to refer to the single-crystal nanoparticle conjugated (also termed herein
  • the coated/encapsulated with the compound defined by A. It can be measured by illuminating the particles with a laser and analysing the intensity fluctuations in the scattered light. Dynamic light scattering measures Brownian motion and relates it to the size of the particles for which light intensity is proportional to the square of the volume of the particle. The resulting diameter corresponds not only to the single-crystal particle diameter, but also to the molecules strongly adsorbed onto its surface. Therefore, the hydrodynamic size is always larger than the size observed by transmission electron microscopy, this later the one of the single-crystal nanoparticles.
  • Typical hydrodynamic diameters of the single-crystal cerium oxide nanoparticles of formula (I) with n higher than 0 and used in the present invention range from 5 to 60 nm, more particularly from 5 to 30 nm. In this particular case, the hydrodynamic diameter is a function of n in formula (I).
  • the combinations encompass one molecule of each type, or several molecules of each type depending of the surface of the nanoparticle directly correlated with its hydrodynamic diameter.
  • the conjugates in particularly those comprising albumin or other proteins adsorbed on the nanoparticle surface, have the property of being captured on the cell surface, mainly due to this so-called "protein corona”. They can so penetrate into the cells, in this case into hepatocytes, and the release of the metal ions performs therein its effects, mainly by scavenging reactive oxygen species (ROS).
  • ROS reactive oxygen species
  • single-crystal cerium oxide nanoparticles of formula (I) for use in the treatment of HCC have a NP with a crystal diameter from 3 to 15 nm.
  • the crystal size diameter of the NP are from 3 to 12 nm, more particularly from 3 to 7 nm, and even more particularly from 4 to 7 nm, more in particular 5 nm. As above indicated when the crystal size diameter of NP is from 3 to 7 nm, then n is from 1 to 12.
  • the single-crystal cerium oxide nanoparticles of formula (I) are in form of a conjugate of formula (I) for being used in the treatment of HCC:
  • NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm;
  • A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof;
  • n is an integer from 1 to 12.
  • NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 24 nm
  • A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and n is an integer from 1 to 40.
  • the single-crystal cerium oxide nanoparticles for use in the treatment of HCC are conjugates.
  • the single-crystal cerium oxide nanoparticle is one in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 12 nm.
  • the single-crystal cerium oxide nanoparticle for use in HCC is one in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm, more particularly from 4 to 5 nm, A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and n is an integer from 1 to 12.
  • the single-crystal cerium oxide nanoparticles are those of formula (I) in the form of conjugates, wherein A is a molecule of albumin, n is and integer from 1 to 40, and NP is a cerium oxide nanoparticle with a crystal size diameter from 3 to 24 nm.
  • NP is a cerium oxide nanoparticle with a crystal size diameter from 3 to 7 nm
  • A is a molecule of albumin
  • n is an integer from 1 to 12.
  • NP is a single-crystal cerium oxide nanoparticle with a crystal size diameter from 4 to 5 nm, an n is from 1 to 4, more particularly, n is 1 .
  • Another particular embodiment of the first aspect of the invention relates to a single-crystal cerium oxide nanoparticle for use in the treatment of HCC, in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 24 nm, A is a molecule of polyvinylpyrrolidone, and n is and integer from 1 to 40.
  • NP in the single-crystal cerium oxide nanoparticle, is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm, and n is an integer from 1 to 12.
  • A is a molecule of polyvinylpyrrolidone; NP is a single-crystal cerium oxide nanoparticle with a crystal size diameter from 4 to 5 nm, and n is from 1 to 4, more particularly, n is 1 .
  • Another particular embodiment of the first aspect of the invention relates to single-crystal cerium oxide nanoparticles of formula (I) for use in the treatment of HCC, in which NP is a cerium oxide nanoparticle with a crystal size diameter from 3 to 7 nm, A is a combination of molecules of albumin and polyvinylpyrrolidone; and n is an integer from 2 to 12, most particularly 2 to 4.
  • the particular conjugates, in which a combination of molecules of albumin and polyvinylpyrrolidone are adsorbed to the NP can be represented as a subgroup of conjugates of formula (I) as follows:
  • A' is polyvinylpyrrolidone;
  • A is albumin; and
  • n and n' are integers from 1 to 2, and particularly n and n' are both 1 .
  • cerium oxide nanoparticles of formula (I) are in particular for use as inhibitors of cell proliferation in hepatocellular carcinoma.
  • the single-crystal cerium oxide nanoparticles of formula (I) are adapted for use as a co-treatment of an hepatocellular carcinoma therapy selected from the group consisting of radiotherapy, microwave ablation (MWA), radiofrequency ablation (RFA), transarterial radioembolization (TARE), chemotherapy, surgical resection, liver transplantation, targeted therapy, hyperthermia and combinations thereof.
  • an hepatocellular carcinoma therapy selected from the group consisting of radiotherapy, microwave ablation (MWA), radiofrequency ablation (RFA), transarterial radioembolization (TARE), chemotherapy, surgical resection, liver transplantation, targeted therapy, hyperthermia and combinations thereof.
  • the single-crystal cerium oxide nanoparticles of formula (I) are adapted for use as a co-treatment of an hepatocellular carcinoma therapy selected from the group consisting of radiotherapy, transarterial
  • TARE radioembolization
  • the single-crystal cerium oxide nanoparticles of formula (I) are for use in the treatment of hepatocellular carcinoma in combination with an hepatocellular carcinoma therapy agent and/or drug, in particular, the agent and/or drug selected from the group consisting of a radiotherapy agent, a chemotherapeutic drug, a targeted therapy drug, and combinations thereof.
  • the single-crystal cerium oxide nanopartides of formula (I) for use in the treatment of HCC are for use in combination with an hepatocellular carcinoma therapy agent and/or drug selected from the group consisting of a radiotherapy agent, a chemotherapeutic drug, a targeted therapy drug, and combinations thereof.
  • an hepatocellular carcinoma therapy agent and/or drug is to be understood as any drug used in this kind of cancer, as well as any compound commonly used in hepatocellular carcinoma therapies, such as the
  • radioenhancement in combination with the radiation itself. It also
  • cerium oxide nanopartides of formula (I) absorb radiation, such as ionizing radiation (including X radiation, gamma rays and higher part of ultraviolet radiation) in a highly efficient manner. The efficiency of this process (for both energy transfer and
  • the single-crystal cerium oxide nanoparticle of formula (I) are for use in the treatment of hepatocellular carcinoma in combination with radiotherapy.
  • the single-crystal cerium oxide nanoparticle of formula (I) are for use in the treatment of hepatocellular carcinoma in combination with radiotherapy.
  • the single-crystal cerium oxide nanoparticle of formula (I) are for use in combination with a targeted therapy agent (also named targeted therapy compound), which can be formulated as that they are for use as co-treatment of a targeted therapy for HCC.
  • a targeted therapy agent also named targeted therapy compound
  • Targeted therapy is defined as the specially targeted delivery vehicles to increase effective levels of chemotherapy for tumor cells while reducing effective levels for other cells. This result in an increased tumor kill and/or reduced toxicity.
  • Particular targeted therapies of HCC include administration of sorafenib. Therefore, the nanoparticles for use according to the invention are, in a particular embodiment for use in
  • CeO2 NPs for use in the treatment of HCC can also be adapted to be used in combination (as co-treatment) with other chemotherapeutic approaches commonly used in this type of cancer and selected from the group consisting of transcatheter arterial chemoembolization (TACE), transcatheter arterial chemotherapy (TAC), intra-arterial infusion chemotherapy (HAC), systemic chemotherapy (SCT), portal vein chemotherapy (PVC), portal vein embolization (PVE), percutaneous acetic acid injection (PAI), percutaneous ethanol injection (PEI), and combinations thereof.
  • TACE transcatheter arterial chemoembolization
  • TAC transcatheter arterial chemotherapy
  • HAC intra-arterial infusion chemotherapy
  • SCT systemic chemotherapy
  • PVC portal vein chemotherapy
  • PVE portal vein embolization
  • PAI percutaneous acetic acid injection
  • PEI percutaneous ethanol injection
  • the single-crystal nanoparticles for use according to the invention are used in combination with one or more of the above therapies, and/or agents for use in these therapies, depending on the stage of the disease (i.e of the HCC).
  • TACE is used in initial stages of HCC and HAC in advanced stages, this later in combination with surgery resection of the tumour and/or liver transplantation.
  • Targeted therapies including the sorafenib are used also in advanced stages of HCC and in combination with surgery resection of the tumour and/or liver transplantation.
  • the invention relates as a second aspect to particular single-crystal cerium oxide nanoparticles of formula (I),
  • NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm;
  • A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof;
  • n is an integer from 1 to 12.
  • Particular embodiments of the second aspect of the invention, thus of the conjugated single-crystal cerium oxide nanoparticles are those in which NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm.
  • A is a molecule of albumin; and n is an integer from 1 to 4, more particularly n is 1 .
  • A is albumin; NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm; and n is an integer from 1 to 4, more particularly n is 1 .
  • the single- crystal cerium oxide nanoparticle is the one of formula (I), wherein A is a molecule of polyvinylpyrrolidone; and n is an integer from 1 to 4, more particularly 1 .
  • A is polyvinylpyrrolidone; NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm; and n is an integer from 1 to 4, more particularly n is 1 .
  • the invention relates also to single-crystal cerium oxide nanoparticles as conjugates of formula (I):
  • A' polyvinylpyrrolidone
  • A is albumin
  • n and n' are integers from 1 to 2, and particularly n and n' are both 1 .
  • PVP has a molecular weight from 10 kDa to 30 kDa, more in particular from 10 kDa to 20 kDa, being preferred PVP of 10 kDa.
  • albumin is mammal albumin, in particular selected from human serum albumin and bovine serum albumin.
  • Human serum albumin is the one defined in in UniProtKB database by accession number P02768, version 255 of the entry and version 2 of the sequence of January 20, 2016.
  • Bovine serum albumin is the one defined in in UniProtKB database by accession number P02769, version 150 of the entry and version 4 of the sequence of December 9, 2015.
  • Single-crystal cerium oxide nanoparticles of formula (I), in which n is zero and NP has a diameter higher than 7, particularly from 8 to 24 nm, are obtainable by a method comprising (a) dissolving a cerium (III) salt, particularly cerium (III) nitrate, in water or in an organic solvent, particularly ethanol; (b) adding an oxidizing and stabilizer compound, in particular selected from
  • TMAOH tetramethylammonium hydroxide and hexamethylenetetramine
  • HMT hexamethylenetetramine
  • the single-crystal cerium oxide nanoparticles of formula (I), in which n is higher than zero, thus the conjugated nanoparticles with n from 1 to 40, are obtainable by a method comprising the steps of:
  • A is albumin, and optionally PVP, then an oxidizing and stabilizer compound is added in this solution 2, in particularly selected from TMAOH and HTM, and preferably TMAOH, and
  • an oxidizing and stabilizer compound is added in this step (iii), in particularly selected from TMAOH and HTM, and preferably TMAOH;
  • CNPs single- crystal CeO2NPs
  • TEM single- crystal CeO2NPs
  • said CNPs being conjugated with albumin, PVP or mixtures thereof, thus being single-crystal cerium oxide nanopartides of formula (I) with n from 1 to 40, and having a hydrodynamic diameter from 10 to 32 nm, in particular from 1 1 to 23 nm, the hydrodynamic diameter measured by DLS.
  • TMAOH is preferred in any of the steps wherein it is used, since it is non-toxic to cells.
  • the cerium (III) salt is cerium (III) nitrate. TMAOH acts as oxidizing agent, as surfactant and also as stabilizer because it avoids nanoparticle aggregation.
  • the buffered solvent in (i) is phosphate- buffered saline (PBS) with a pH from 6.0 to 8.5 that comprises water and salts selected from sodium hydrogenphosphate, sodium chloride and mixtures thereof, and optionally potassium chloride and potassium
  • PBS phosphate- buffered saline
  • solution 2 has a pH below 9, preferably from 6.0 to 8.5, more preferably from 7.0 to 8.0.
  • the stabilizer for colloidal stability and to avoid CeO2NPs aggregation has to be administrated before aggregation takes place, which is immediately after nucleation, this is, at the beginning of the synthesis process.
  • other stabilizers like TMAOH can be added during synthesis.
  • the cerium (III) salt is particularly cerium (III) nitrate, and in yet another more particular embodiment it is in a concentration at solution 2 to give an initial cerium (III) concentration from 9.0 mM to 10.0 mM.
  • the final concentration of albumin in solution 1 is from 0.70 to 0.80 mM, more particularly 0.75 mM.
  • nanopartides of formula (I) of the invention wherein NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm; A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and n is an integer from 1 to 12, are obtainable by a method comprising the steps of:
  • A is albumin, and optionally PVP, then an oxidizing and stabilizer compound is added in this solution 2, in particularly selected from TMAOH and HTM, and preferably TMAOH, and
  • an oxidizing and stabilizer compound is added in this step (iii), in particularly selected from TMAOH and HTM, and preferably TMAOH;
  • CNPs single- crystal CeO 2 NPs
  • TEM crystal size diameter measured by TEM from 3 to 7 nm
  • said CNPs being conjugated with albumin, PVP or mixtures thereof, thus being single-crystal cerium oxide nanopartides of formula (I) with n from 1 to 12, and having a hydrodynamic diameter from 1 1 to 23 nm, the hydrodynamic diameter measured by DLS.
  • the nanoconjugates are constituted by single-crystal nanopartides, duly conjugated with albumin, PVP or both.
  • these single-crystal cerium oxide nanopartides of formula (I), in which n is higher than zero, thus the conjugated nanopartides with n from 1 to 40, and obtainable as indicated in any of the above embodiments, are catalytically active single- crystal cerium oxide nanopartides.
  • the nanopartides being catalytically active means that they retain its superoxide dismutase (SOD) and catalase mimetic activity.
  • This catalytically active feature can be measured by many technologies, such as for example, by means of the CeO2NPs antioxidant effect on fluorescently-labeled hydrogen peroxidase (EuTc-H2O2 complex), Amplex Red or 2', 7' -dichlorofluorescin diacetate (DCFDA).
  • Particular pharmaceutical or veterinary compositions comprising the nanopartides as defined above, are those further comprising physiologic saline buffer, such as Phosphate-buffered saline (PBS).
  • PBS Phosphate-buffered saline
  • physiologic saline buffer is to be understood a water-based salt solution with osmolarity, pH (6- 7.5) and ion concentrations of mammal bodies, in particular of humans.
  • compositions comprise the serum of the individual mammal to which the composition is to be administered.
  • HR-TEM Transmission Electron Microscopy
  • FEI Tecnai G2 F20 200 kV The ultrafin 200-mesh copped TEM grids (Ted-Pella, Inc.) were placed on a filter paper. Afterwards, fifty microliters of the NPs colloidal suspension were deposited drop by drop over the grid and left to dry in air. The obtained HR- TEM images were analyzed by Image J program: for each sample, at least 150 NPs were measured and the average size and size-distribution were obtained.
  • UV-Vis Spectroscopy UV-visible spectra was acqired with Schimadzu UV-2400 spectrofotometer. Regarding the detection limits of the apparatus and a high concentration of the synthesis, an aliquot of the synthesis solution was diluted in water (1/15) and than measured in a wavelength range between 260 and 800 nm.
  • DLS A 1 ml aliquot of the synthesis solution was placed in a sizing couvette and analyzed by a Zetasizer Nano-ZS (Malvern Instruments). Thus, the surface charge (Zeta Potential, ZP), the Dynamic Light Scattering (DLS) and the Isoelectric Point (pi) of the nanoparticles were measured.
  • X-rav diffraction A 10 ml aliquot of the synthesis solution was centrifuged twice (45 min at 12.000g), in order to ensure a maximal precipitation of the nanoparticles. A supernatant was discarded and the precipitates were dried at room
  • Example 1 Synthesis of cerium oxide nanoparticles.
  • LPS lipopolysaccharide
  • FIG. 3 (A) shows pH evolution of the synthesis solution
  • Nanoceria characterization by Electron Microscopy To minutely examine the quality of produced CeO 2 NPs, in terms of
  • FIG. 4 shows as-synthesized PVP- coated samples
  • FIG. 4 (b) shows the dark-field STEM-HAADF image of the NPs
  • FIG. 4 (c) inspects the homogeneity
  • FIG. 4 (d) shows a typical single-crystalline CeO 2 NP and its correspondent Fourier Transformation, FFT (FIG. 4 (e)).
  • FFT Fourier Transformation
  • the structural and compositional characterization, phase identification and size of CeO 2 NPs were analyzed by X-ray Diffraction (XRD).
  • XRD X-ray Diffraction
  • the XRD pattern of as-synthesized CeO 2 NPs of 4 nm is displayed in FIG. 5.
  • the XRD pattern was scanned from 20 to 80 degrees and the XRD profile confirmed the monocrystalline nature of CeO 2 NPs (single-crystal cerium oxide
  • Crystal size diameter estimation Three techniques were used to estimate the average crystal size diameter of as-synthesized in 1 .1 or 1 .2 CeO 2 NPs: Image J analysis software for HR- TEM; size estimation using the Scherrer equation of XRD pattern and the Dynamic Light Scattering (DLS) of colloidal suspension of the NPs (measuring hydrodynamic diameter). Table 1 shows a comparison between three sizing techniques for as-synthesized CeO2NPs: (a) Dynamic Light Scattering (DLS) and the polydispersity index (PDI) of this DLS, (b) Image J analysis of HR- TEM images, and (c) Scherrer equation calculations from XRD profile.
  • DLS Dynamic Light Scattering
  • PDI polydispersity index
  • PDI is a parameter in DLS related with size distribution of molecules or particles in suspension. For a perfectly uniform sample, the PDI would be 0.0. PDI from 0.0 to 0.1 reflects a narrow non-uniform distribution type sample. From 0.1 to 0.4 the PDI indicates a moderate non-uniform distribution type sample. PDI higher than 0.4 is usually understood as a broad distribution type sample.
  • the size (diameter in nm) and aggregation state of the CeO 2 NPs conjugated with albumin disclosed in 1 .2 were again corroborated by DLS technique. In both measurements, by intensity and by number, it was observed one single peak at around 12 nm (hydrodynamic diameter). The absence of peaks at larger size axis corroborates the absence of aggregates in the designed synthesis method. Moreover, as an internal control, the BSA-coated NPs were purified by centrifugation and measured by DLS the supernatant only (FIG. 6 (c) and (d)), that contains free BSA in excess. The size distribution coincides with previously obtained data in FIG.
  • DEN diethylnitrosamine
  • liver sections of non-treated with CeO2 NPs rats had a more dysmorphic appearance also with more superficial deformities than liver of CeO2 NPs treated rats.
  • the liver weight percentage in relation to total body weight was slightly lower in animals receiving CeO2 NPs in comparison with non-treated animals. Non meaningful differences were observed in serologic standard hepatic functional parameters and in renal parameters between bot animal groups.
  • Data are depicted in FIG. 1 (A and B), wherein the microscope image (FIG. 1 (A)) of the liver of non- treated cells (VEHICLE) shows a higher percentage of Ki-67 stained
  • Example 3 CeO2 NPs for use in combination with radiotherapy.
  • FIG. 7 is an image of X ray contrast imaging of samples at different CeO2NPs concentrations ranging from 0 to 100 mg/ml .
  • the samples where imaged at 90 kV and 160 microA.
  • the bright white signal, indicative of X-ray absorption increases with CeO2 concentration as expected. This X-ray absorption is at the basis of X-ray imaging and X-ray therapy.
  • CeO2 NPs absorbed X radiation, in a highly efficient manner. Transfer of energy was also high with these nanoparticles. Therefore, they are good contrast agents (in particular as X-ray contrast agents), as well as good to be used in combination with radiotherapy, providing to the subject the intrinsically effect of the ceria nanoparticles (due to their superoxide dismutase (SOD) and catalase mimetic activity), and enhancing the effect of the administered radiotherapy.
  • SOD superoxide dismutase
  • catalase mimetic activity enhancing the effect of the administered radiotherapy.
  • Example 4 Catalytic activity of PVP-conjugated CeO 2 NPs (single-crystal cerium oxide nanoparticles of formula (I) in which A is PVP)
  • Results are depicted in FIG. 8, a graphic displaying for each assayed nanoparticle type the decay of fluorescence (F) versus time (t, in min).
  • PVP-coating slightly decreases CeO 2 NPs reactivity, but as deducible from FIG. 8, PVP-coated 5nm-sized CeO 2 NPs of the invention maintained the catalytic activity, and not so did commercial.

Abstract

The invention relates to cerium oxide nanoparticles useful in the treatment of hepatocellular carcinoma (HCC), said nanoparticles being cerium oxide single-crystalline nanoparticles of formula (I) NP-(A)n (I), wherein NP is a single-crystalline cerium oxide nanoparticle with a crystal diameter from 3 to 24 nm; A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and n is an integer from 0 to 40, and wherein if the crystal diameter of the nanoparticle is from 3 to 7 nm, then n is an integer from 1 to 12. The invention proposes also particular nanoparticles of formula (I) as conjugates, as well as pharmaceutical compositions comprising them.

Description

Ceria nanoparticles for use in the treatment of hepatocellular carcinoma
This application claims the benefit of European Patent Application 16163838.2 filed April 5, 2016.
The present invention relates to the field of medicine, in particular to cancer treatment. It also relates to nanoparticles and conjugates comprising them, as well as to pharmaceutically or veterinary compositions for the treatment of cancer.
BACKGROUND ART
Liver cancer is a major health problem for being the second cause of cancer death worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Recommended treatments relate to surgical resection, liver transplantation and local ablation. In addition, Sorafenib, a receptor tyrosine kinase inhibitor, may be used in patients with advanced hepatocellular carcinoma. Sorafenib is a small molecule that inhibits tumor-cell proliferation and tumor angiogenesis.
Recently, nanomaterials have been proposed for use in the treatment of certain cancers. As a way of example, d extra n -coated cerium oxide
nanoparticles have been administered to an A375 xenograft model of nude mice in order to test them in a melanoma model. The data are summarized in ANN et al., "Downregulation of Tumor Growth and Invasion by Redox-Active
Nanoparticles", Antioxidant & Redox Signaling (Original Research
Communication) - 2013, vol. 00, pp.: 1 -14 (DOI: 10.1089/ars.2012.4831 ). ANN et al. concluded that redox-active cerium oxide nanoparticles (CNPs) had selective pro-oxidative and antioxidative properties and could be the basis of a new paradigm in the treatment and prevention of cancer. Dextran -coated cerium oxide nanoparticles implied moreover the advantage of avoiding agglomeration when the individual nanoparticles were of the size 3-5 nm.
Cerium oxide nanoparticles (CNPs) have emerged thus in biomedical applications due to their superoxide dismutase (SOD) and catalase mimetic activity. CNPs act as catalysts with mixed valence that can exist in a reduced (+3) or oxidized (+4) state. However, tests performed in human hepatoma cells have demonstrated that cerium oxide nanopartides are toxic. As an example, Cheng et al., in "Cerium oxide nanopartides induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signalling pathways",
Toxicology in vitro - 2013, vol. no. 27, pp.: 1082-1088, demonstrated that CeO2 nanopartides, widely used in many industrial fields, promoted a reduced viability, a dramatic morphological damage and induced apoptosis in SMMC- 7721 cells. The nanopartides used by Cheng et al. were hexahedral CeO2 nanopartides with a primary particle diameter of 20-30 nm. Sizes of particles higher than 25 nm tend to accumulate in tissue and they exert there toxic effects. In addition, it is known that high particle diameters may even not be catalytic, thus ineffective. On the other side, CNPs, or any other nanopartides with low diameters (fewer than 7 nm) have low residence time in the body, and poor solubility (See Hak Soo et al., "Renal clearance of quantum dots", Nature biotechnology - 2007, vol. no. 25(10), pp.: 1 165-1 170). Besides, during the preparation of CNPs toxic reagents are employed (such as hexamethylenetetramine; HMT), making them hazardous for biomedical applications if they are not separated from the nanopartides before, which sometimes is difficult or imply expensive procedures.
Thus, being still a need to find alternative therapeutic approaches for HCC, it is also a need to find compounds with the beneficial effects of nanopartides in cancer, but avoiding the problems associated with its administration, mainly due to agglomeration and low residence time in the body.
SUMMARY OF THE INVENTION
Inventors propose using particular cerium oxide nanopartides in the treatment of HCC, which surprisingly allowed increasing the survival of treated animals in a remarkably way. Moreover, proliferation of cancer cells was highly reduced when the nanopartides were administered to mammals with induced HCC. Thus, the treatment avoided tumour growth.
A first aspect the invention relates to single-crystal cerium oxide nanopartides of formula (I) for use in the treatment of hepatocellular carcinoma, formula (I) defined by:
NP-(A)n (I), wherein
NP is a cerium oxide nanoparticle with a crystal diameter measured by transmission electron microscopy (TEM) from 3 to 24 nm;
A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone (PVP), and combinations thereof; and
n is an integer from 0 to 40,
and wherein if the crystal diameter of the nanoparticle is from 3 to 7 nm, then n is an integer from 1 to 12.
When n is higher than 0, this means that the nanoparticles are conjugated with A, so that they are also called in the present invention as conjugates.
Although other cancers have been treated with cerium oxide nanoparticles (see ANN et al., supra) effectivity cannot be extrapolated to other cancer types and less in liver cancer, where in vitro data have correlated with high toxic effects (see Cheng et al., supra). Thus, the invention provides the unexpected effect of conjugates with CNPs or of CNPs that can be safely applied to HCC treatment. The conjugates or CNPs are effective due to a proper non- agglomeration (or non-aggregation) of the nanoparticles, and due to a size that, though relative small, can be retained in liver.
In a second aspect, the invention relates to particular single-crystal cerium oxide nanoparticles of formula (I),
NP-(A)n (I), wherein
NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm;
A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and
n is an integer from 1 to 12.
These particular single-crystal cerium oxide nanoparticles of formula (I) are herewith also named conjugates as above indicated, since n is always an integer higher than 0, namely from 1 to 40 nm.
As exposed before, also these particular conjugates imply the advantages of being effective against liver cancer cells, since despite being small (crystal diameter size of 3-7 nm) and without agglomerating, they are retained in the liver. Once retained, they can exert its function, because they maintain its catalytic function against reactive oxygen species (ROS). In addition they are not toxic at effective doses.
Yet another aspect of the invention is a conjugated single-crystal cerium oxide nanoparticle of formula (I):
NP-(A)n (I), wherein
NP is a cerium oxide nanoparticle with a crystal diameter from 8 to 24 nm;
A is a molecule of albumin; and
N is an integer from 5 to 40.
These later albumin conjugates with CNPs of formula (I) do not aggregate, and due to its size they are retained in the liver, thus performing their action.
Some authors have reported recently the effects of cerium oxide encapsulated albumin nanoparticles. At this respect it is mentioned the document of Bushan et al., "Antioxidant nanozyme: a facile synthesis and evaluation of the reactive oxygen species scavenging potential of nanoceria encapsulated albumin nanoparticles", Journal of Materials Chemistry B - 2015, vol. no. 3, pp.: 4843- 4852. Encapsulation of CNPs (diameter of 4.35 ± 1 .07 nm, measured with TEM) in albumin nanoparticles did not alter CNPs functionality in terms of protecting cells from entering oxidative stress (from gene expression studies in L-132 cell line of cervix). The cells were viable (assay performed with MTT dye); and the cellular uptake of CNPs was found to be increased with encapsulated CNPs in a time dependent manner. CNPs of Bushan et al. were synthesized by the hydrothermal method. By TEM images it was seen that the average size of CNPs was of 4.35 ± 1 .07 nm. CNPs were then encapsulated inside albumin nanoparticles via a desolvation technique using ethanol as the desolvating agent and glutaraldehyde as a cross linking agent to obtain least aggregated spherical BCNPs with uniform distribution.
The so synthesized BCNPs allowed concluding that a novel artificial antioxidant encapsulated delivery system had been developed, but the data from Bushan et al. are not conclusive for cancer treatment. In addition, these ceria encapsulated albumin nanoparticles are different from conjugates of formula (I) of present invention. Another study showing how bovine serum albumin (BSA) is adsorbed to CNPs is derivable from Marsalek et al., "Adsoprtion of Bovine Serum Albumin on CeO2", International Journal of Chemical, Nuclear, Materials and Metallurgical Engineering -2014, Vol. no. 8(12), pp.: 1269-1272. In this document CNPs are formed and then BSA is adsorbed by means of a vigorous stirring, sonication and further centrifugation. Authors conclude that BSA is preferentially adsorbed onto positively charged surface of the CNPs. With the methodology used by Marsalek et al., it is post-synthesis of the nanoparticles that they are coated with BSA. This procedure has nothing to do with avoiding aggregation of the nanoparticles. Besides, in many of the by Marsalek's disclosed conditions BSA is denaturalized.
The single-crystal cerium oxide nanoparticles of the invention can be administered to mammals in several forms appropriated for administration. Thus, another aspect of the invention is a pharmaceutical or veterinary composition comprising these nanoparticles of formula (I) as defined above, together with one or more pharmaceutically or veterinary acceptable excipients or carriers. These pharmaceutical or veterinary compositions comprising the new single-crystal cerium oxide nanoparticles of the invention are also for use in the treatment of hepatocellular carcinoma.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 , related with Example 2, shows in (A) optical microscope images (200X) of Ki-67-tincted hepatic cells from Wistar rats treated with cerium oxide nanoparticles (CeO2NPs) or with vehicle, which was a suspension of TMAOH 0.8 mM in phosphate saline buffer (PBS). In FIG. 1 (B) the percentage of Ki-67 nuclei hepatocytes per animal are shown for each group. FIG. 2, related with Example 2, is a graphic with the survival (percentage of survival; %S) of the rats versus the time (T, weeks (W)) after reception of last dose of single-crystal cerium oxide nanoparticles of formula (I) in which A is albumin (CeO2NPs) or of vehicle as disclosed for FIG. 1 .
FIG. 3, related with Example 1 depicts the monitoring of the synthesis kinetics of CeO2NPs (single-crystal cerium oxide nanoparticles of formula (I) in which A is either albumin or PVP). In FIG. 3 (A) the pH evolution of the synthesis solution. In FIG. 3 (B) UV-visible spectrum of initial cerium (III) precursor (black line); as-synthesized NPs at 1 h of reaction, diluted 1 /20 (soft grey line); as-synthesized NPs at 48h of reaction, diluted 1 /20 (dark grey line). Time in minutes (min), pH, means initial pH and pHf means final pH; UV-vis Abs is the absorption in arbitrary units (u.a); and the wavelength is in nanometers (nm).
FIG. 4, also related with Example 1 , shows the characterization of PVP- conjugated CeO2NPs (single-crystal cerium oxide nanoparticles of formula (I) in which A is PVP) by transmission electron microscopy (TEM). In FIG. 4 a) high resolution TEM micrograph (HR-TEM) at high (400.000X) magnification, revealing the atomic planes of single-crystal nanoparticles; in FIG. 4 b) High Angle Annular Dark Field-Scanning Transmission Electron Microscopy (STEM-HAADF) image; in FIG. 4 c) HR-TEM image at low magnification (7.000 check); in FIG. 4 d) atomic resolution HR-TEM image of one single (single-crystal) CeO2NP, showing spherical morphology and the correspondent atomic planes; in FIG. 4 e) Fast Fourier Transformation (FFT) digital diffractogram calculated from the particle shown in FIG. 4 d); in FIG. 4 f) Electron energy-loss spectroscopy (EELS, X-axis Energy in kiloelectrovolts (keV); Y-axis counts (C) in arbitrary units (a.u.)) map of the chemical composition of as synthesized CeO2NP. Panels d), e) and F appear in Cont.FIG. 4 as a magnified image of squared single-crystal nanoparticle of panel c) in FIG. 4.
FIG. 5, related with Example 1 , is an X-ray diffraction (XRD) pattern of a single-crystal CeO2 nanoparticle of formula (I) with 5 nm of crystal size diameter.
FIG. 6, related with Example 1 , depicts the Dynamic Light Scattering (DLS) of BSA-coated CeO2NPs of the invention (single-crystal cerium oxide nanoparticles of formula (I) in which A is albumin). In FIG. 6 a) DLS of the as- synthesized BSA-coated (1 mM) CeO2NPs, by Intensity. In FIG. 6 b) DLS of the as-synthesized BSA-coated (1 mM) CeO2NPs, by Number. In FIG. 6 c) DLS of the supernatant only, containing free BSA, by Intensity. In FIG. 6 d) DLS of the supernatant only, containing free BSA, by Number. In X-axis the size (hydrodynamic diameter in nm; d, nm. Y-axis shows percentage (Percent) of Intensity (I) or the percentage of Number (N). FIG. 7 is an image of X ray contrast imaging of samples at different CeO2NPs concentrations ranging from 0 to 100 mg/ml .
FIG. 8 is a graphic showing the CeO2NPs antioxidant effect on fluorescently- labeled hydrogen peroxidase (EuTc-H2O2 complex), (a) Commercial 25-nm- sized CeO2NPs; (b) PVP-coated 5 nm-sized CeO2NPs (PVP-conjugated CeO2NPs of formula (I) in which A is PVP); (c) surfactant-free mix of CeO2NPs (mix of sizes from 6 to dozens of nm; mix, naked); (d) surfactant-free 5-nm- sized CeO2NPs (naked). (F) is fluorescence and (t) time in minutes(min).
DETAILED DESCRIPTION OF THE INVENTION
All terms as used herein, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the description and claims unless an otherwise expressly set out definition provides a broader definition.
The term "nanoconjugate" refers to a single-crystal cerium oxide nanoparticle which is attached to another compound selected from polyvinylpyrrolidone and proteins, in particular globular proteins, such as albumin. In particular, the term conjugate, also referred to as NP-A, refers to a single-crystal cerium oxide nanoparticle (NP) which is adsorbed by coordinate bonding to a molecule (A), that can be PVP and/or albumin. By "single-crystal cerium oxide nanoparticle" it is to be understood that said nanoparticle is not aggregated (by weak physical interaction) with other nanoparticles, in such a way that the nanoconjugate comprises only one nanoparticle as a core of the conjugate and of a particular crystal size diameter (or simply crystal diameter). All these structures can be seen by X-Ray diffraction and TEM. Thus, single-crystal or monocrystal means that there is only one crystal of cerium oxide configuring the nanopartide, with a particular crystal diameter.
The term "nanopartide" as used herein, refers to a particle with at least two dimensions at the nanoscale, particularly with all three dimensions at the nanoscale, where the nanoscale is the range about 1 nm to about 100 nm, more particularly from 1 to 50 nm, and even more particularly from 5 to 20 nm. Preferred crystal diameters of the nanopartides of the invention are from 5 to 10 nm (including, 5, 6, 7, 8, 9, and 10 nm), more preferably 5 nm. As regards the shape of the nanopartides described herein, there are included spheres and polyhedral. In a particular embodiment the nanopartide is spherical.
Particularly, when the nanopartide is substantially rod-shaped with a substantially circular cross-section, such as a nanowire or a nanotube, the "nanopartide" refers to a particle with at least two dimensions at the
nanoscale, this two dimensions being the cross-section of the nanopartide.
As used herein, the term "size" refers to a characteristic physical dimension. For example, in the case of a nanopartide that is substantially spherical, the size of the nanopartide corresponds to the diameter of the nanopartide. When referring to a set of nanopartides as being of a particular size, it is
contemplated that the set of nanopartides can have a distribution of sizes around the specified size. Thus, as used herein, a size of a set of
nanopartides can refer to a mode of a distribution of sizes, such as a peak size of the distribution of sizes. In addition, when not perfectly spherical, the diameter is the equivalent diameter of the spherical body including the object.
The term "cerium oxide" or "ceria" refers to cerium (III) oxide (Ce3+) and cerium (IV) oxide (Ce4+) species that are both present when constituting the nanopartides. Although many of the cerium oxide nanopartides are usually in the (Ce4+) oxidation state, small cerium oxide nanopartides are also in the (Ce3+) oxidation state. Cerium oxide nanopartides (abbreviated also as Ceria- NP, CeO2-NP or simply CNP) are used in a variety of applications mainly due to its high surface area and the ability of cerium oxide to cycle between (III and IV) oxidation states. In a particular embodiment, cerium oxide
nanopartides are in the (Ce4+) oxidation state When in this description the diameter of the nanoparticle is mentioned, it relates to the crystal size diameter (single-crystal). Crystal size is usually measured from X-ray diffraction patterns while particle size is measured by TEM. In the case of single-crystal nanoparticles, XRD and TEM sizes coincide. Typical crystal size diameters of the nanoparticles used in the present invention range from 3 to 24 nm (including 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 and 24 nm), when measured by TEM, more particularly the crystal size diameter of the nanoparticles is about 5 nm.
The "hydrodynamic diameter" determined by dynamic light scattering (DLS) techniques based on the Stoke-Einstein equation is used herewith to refer to the single-crystal nanoparticle conjugated (also termed herein
coated/encapsulated) with the compound defined by A. It can be measured by illuminating the particles with a laser and analysing the intensity fluctuations in the scattered light. Dynamic light scattering measures Brownian motion and relates it to the size of the particles for which light intensity is proportional to the square of the volume of the particle. The resulting diameter corresponds not only to the single-crystal particle diameter, but also to the molecules strongly adsorbed onto its surface. Therefore, the hydrodynamic size is always larger than the size observed by transmission electron microscopy, this later the one of the single-crystal nanoparticles. Typical hydrodynamic diameters of the single-crystal cerium oxide nanoparticles of formula (I) with n higher than 0 and used in the present invention range from 5 to 60 nm, more particularly from 5 to 30 nm. In this particular case, the hydrodynamic diameter is a function of n in formula (I).
Finally, when in the present invention it appears the expression "combinations thereof, it relates to the combination of molecules of albumin and
polyvinylpyrrolidone in the conjugated cerium oxide nanoparticles of formula (I), each of them adsorbed on the nanoparticle surface in order to make the nanoparticles compatible for the body (biocompatible). The combinations encompass one molecule of each type, or several molecules of each type depending of the surface of the nanoparticle directly correlated with its hydrodynamic diameter. The conjugates, in particularly those comprising albumin or other proteins adsorbed on the nanoparticle surface, have the property of being captured on the cell surface, mainly due to this so-called "protein corona". They can so penetrate into the cells, in this case into hepatocytes, and the release of the metal ions performs therein its effects, mainly by scavenging reactive oxygen species (ROS).
In a particular embodiment of the first aspect of the invention, single-crystal cerium oxide nanoparticles of formula (I) for use in the treatment of HCC have a NP with a crystal diameter from 3 to 15 nm. In yet another particular embodiment the crystal size diameter of the NP are from 3 to 12 nm, more particularly from 3 to 7 nm, and even more particularly from 4 to 7 nm, more in particular 5 nm. As above indicated when the crystal size diameter of NP is from 3 to 7 nm, then n is from 1 to 12.
In this particular case, wherein the crystal size diameter of the NP is from 3 to 7 nm, it is also said that the single-crystal cerium oxide nanoparticles of formula (I) are in form of a conjugate of formula (I) for being used in the treatment of HCC:
NP-(A)n (I), wherein
NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm;
A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and
n is an integer from 1 to 12.
On the other side, when crystal size diameters of NP in formula (I) are higher than 7 nm, these single-crystal cerium oxide nanoparticles of formula (I) can be used without being conjugated to albumin and/or PVP, thus n being an integer with value 0. The reason for that higher than 7 nm crystal size diameter, is to avoid fast renal clearance of the nanoparticles.
In another particular embodiment of the first aspect, optionally in combination with any embodiment above or below, NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 24 nm; A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and n is an integer from 1 to 40. In this particular embodiment, the single-crystal cerium oxide nanoparticles for use in the treatment of HCC are conjugates.
In yet another particular embodiment of the first aspect, the single-crystal cerium oxide nanoparticle is one in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 12 nm.
In another particular embodiment, the single-crystal cerium oxide nanoparticle for use in HCC, is one in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm, more particularly from 4 to 5 nm, A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and n is an integer from 1 to 12.
In another more particular embodiment of the first aspect, optionally in combination with the embodiments above or below, the single-crystal cerium oxide nanoparticles are those of formula (I) in the form of conjugates, wherein A is a molecule of albumin, n is and integer from 1 to 40, and NP is a cerium oxide nanoparticle with a crystal size diameter from 3 to 24 nm.
Another particular embodiment of the first aspect relates to a single-crystal cerium oxide nanoparticle for use in the treatment of HCC, in which NP is a cerium oxide nanoparticle with a crystal size diameter from 3 to 7 nm, A is a molecule of albumin; and n is an integer from 1 to 12. In yet another more particular embodiment, NP is a single-crystal cerium oxide nanoparticle with a crystal size diameter from 4 to 5 nm, an n is from 1 to 4, more particularly, n is 1 .
Another particular embodiment of the first aspect of the invention relates to a single-crystal cerium oxide nanoparticle for use in the treatment of HCC, in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 24 nm, A is a molecule of polyvinylpyrrolidone, and n is and integer from 1 to 40.
In another more particular embodiment, in the single-crystal cerium oxide nanoparticle, NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm, and n is an integer from 1 to 12. In yet a more particular embodiment, A is a molecule of polyvinylpyrrolidone; NP is a single-crystal cerium oxide nanoparticle with a crystal size diameter from 4 to 5 nm, and n is from 1 to 4, more particularly, n is 1 . Another particular embodiment of the first aspect of the invention relates to single-crystal cerium oxide nanoparticles of formula (I) for use in the treatment of HCC, in which NP is a cerium oxide nanoparticle with a crystal size diameter from 3 to 7 nm, A is a combination of molecules of albumin and polyvinylpyrrolidone; and n is an integer from 2 to 12, most particularly 2 to 4. The particular conjugates, in which a combination of molecules of albumin and polyvinylpyrrolidone are adsorbed to the NP can be represented as a subgroup of conjugates of formula (I) as follows:
Figure imgf000013_0001
A' is polyvinylpyrrolidone; A is albumin; and n and n' are integers from 1 to 2, and particularly n and n' are both 1 .
All these above disclosed cerium oxide nanoparticles of formula (I) are in particular for use as inhibitors of cell proliferation in hepatocellular carcinoma.
In yet another particular embodiment of the first aspect of the invention, the single-crystal cerium oxide nanoparticles of formula (I) are adapted for use as a co-treatment of an hepatocellular carcinoma therapy selected from the group consisting of radiotherapy, microwave ablation (MWA), radiofrequency ablation (RFA), transarterial radioembolization (TARE), chemotherapy, surgical resection, liver transplantation, targeted therapy, hyperthermia and combinations thereof.
More in particular, the single-crystal cerium oxide nanoparticles of formula (I) are adapted for use as a co-treatment of an hepatocellular carcinoma therapy selected from the group consisting of radiotherapy, transarterial
radioembolization (TARE), chemotherapy, targeted therapy, hyperthermia and combinations thereof.
As "co-treatment" is to be understood that the nanoparticles are for
simultaneous, separate or sequential use with other HCC therapies.
Thus, the single-crystal cerium oxide nanoparticles of formula (I) are for use in the treatment of hepatocellular carcinoma in combination with an hepatocellular carcinoma therapy agent and/or drug, in particular, the agent and/or drug selected from the group consisting of a radiotherapy agent, a chemotherapeutic drug, a targeted therapy drug, and combinations thereof. This means that the single-crystal cerium oxide nanopartides of formula (I) for use in the treatment of HCC, are for use in combination with an hepatocellular carcinoma therapy agent and/or drug selected from the group consisting of a radiotherapy agent, a chemotherapeutic drug, a targeted therapy drug, and combinations thereof.
As "an hepatocellular carcinoma therapy agent and/or drug" is to be understood as any drug used in this kind of cancer, as well as any compound commonly used in hepatocellular carcinoma therapies, such as the
compounds used in radiotherapy for promoting radiosensitation and
radioenhancement in combination with the radiation itself. It also
encompasses agents used in transcatheter arterial chemoembolization (TACE), heat sensitive compounds (i.e, liposomes) for use in radiofrequency ablation (RFA),as well as drugs (i.e sorafenib) for a targeted therapy. As will be shown in the examples below, cerium oxide nanopartides of formula (I) absorb radiation, such as ionizing radiation (including X radiation, gamma rays and higher part of ultraviolet radiation) in a highly efficient manner. The efficiency of this process (for both energy transfer and
biodistribution) is mainly due to heavy atoms nanopartides with small size and to the fact that they are not aggregated (but single-crystals). For the same reason, transfer of energy is also high with these nanopartides. All these properties make them good as contrast agents (in particular as X-ray contrast agents), as well as good in radiotherapy (radiosensitation and
radioenhancement), since effect of radiation is enhanced. Thus in a more particular embodiment, the single-crystal cerium oxide nanoparticle of formula
(I) are for use in the treatment of hepatocellular carcinoma in combination with a radiotherapy agent. Thus, the single-crystal cerium oxide nanoparticle of formula (I) are for use in the treatment of hepatocellular carcinoma in combination with radiotherapy.
Further, the single-crystal cerium oxide nanoparticle of formula (I) are for use in combination with a targeted therapy agent (also named targeted therapy compound), which can be formulated as that they are for use as co-treatment of a targeted therapy for HCC. Targeted therapy is defined as the specially targeted delivery vehicles to increase effective levels of chemotherapy for tumor cells while reducing effective levels for other cells. This result in an increased tumor kill and/or reduced toxicity. Particular targeted therapies of HCC include administration of sorafenib. Therefore, the nanoparticles for use according to the invention are, in a particular embodiment for use in
combination with sorafenib, particularly in advanced stages of the disease, and optionally with surgical resection. In addition, CeO2 NPs for use in the treatment of HCC can also be adapted to be used in combination (as co-treatment) with other chemotherapeutic approaches commonly used in this type of cancer and selected from the group consisting of transcatheter arterial chemoembolization (TACE), transcatheter arterial chemotherapy (TAC), intra-arterial infusion chemotherapy (HAC), systemic chemotherapy (SCT), portal vein chemotherapy (PVC), portal vein embolization (PVE), percutaneous acetic acid injection (PAI), percutaneous ethanol injection (PEI), and combinations thereof.
The single-crystal nanoparticles for use according to the invention are used in combination with one or more of the above therapies, and/or agents for use in these therapies, depending on the stage of the disease (i.e of the HCC). Thus, for example TACE is used in initial stages of HCC and HAC in advanced stages, this later in combination with surgery resection of the tumour and/or liver transplantation. Targeted therapies including the sorafenib are used also in advanced stages of HCC and in combination with surgery resection of the tumour and/or liver transplantation.
The invention relates as a second aspect to particular single-crystal cerium oxide nanoparticles of formula (I),
NP-(A) ',n (I), wherein
NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm;
A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and
n is an integer from 1 to 12. Particular embodiments of the second aspect of the invention, thus of the conjugated single-crystal cerium oxide nanoparticles are those in which NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm.
In a more particular embodiment of the second aspect of the invention, optionally in combination with any embodiment above or below, in the single- crystal cerium oxide nanoparticle, A is a molecule of albumin; and n is an integer from 1 to 4, more particularly n is 1 . Most particularly, A is albumin; NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm; and n is an integer from 1 to 4, more particularly n is 1 .
In another more particular embodiment of the second aspect of the invention, optionally in combination with any embodiment above or below the single- crystal cerium oxide nanoparticle is the one of formula (I), wherein A is a molecule of polyvinylpyrrolidone; and n is an integer from 1 to 4, more particularly 1 . Most particularly, A is polyvinylpyrrolidone; NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm; and n is an integer from 1 to 4, more particularly n is 1 .
The invention relates also to single-crystal cerium oxide nanoparticles as conjugates of formula (I):
(A')n-NP-(A)n (I) wherein A' is polyvinylpyrrolidone; A is albumin; and n and n' are integers from 1 to 2, and particularly n and n' are both 1 .
In a particular embodiment of any of the first and second aspects of the invention, optionally in combination with any of the embodiments, PVP has a molecular weight from 10 kDa to 30 kDa, more in particular from 10 kDa to 20 kDa, being preferred PVP of 10 kDa.
In another particular embodiment of any of the aspects of the invention, optionally in combination with any of the embodiments, albumin is mammal albumin, in particular selected from human serum albumin and bovine serum albumin. Human serum albumin is the one defined in in UniProtKB database by accession number P02768, version 255 of the entry and version 2 of the sequence of January 20, 2016. Bovine serum albumin is the one defined in in UniProtKB database by accession number P02769, version 150 of the entry and version 4 of the sequence of December 9, 2015.
Single-crystal cerium oxide nanoparticles of formula (I), in which n is zero and NP has a diameter higher than 7, particularly from 8 to 24 nm, are obtainable by a method comprising (a) dissolving a cerium (III) salt, particularly cerium (III) nitrate, in water or in an organic solvent, particularly ethanol; (b) adding an oxidizing and stabilizer compound, in particular selected from
tetramethylammonium (TMAOH) hydroxide and hexamethylenetetramine (HMT) in a ratio of volume of solution (a) per volume of oxidizing and stabilizer compound from 95/5 (v/v) to 80/20 (v/v), more in particular of 90/10 (v/v); and (c) optionally centrifuging and resuspending the nanoparticles in aqueous solution, said aqueous solution also optionally comprising TMAOH as stabilizer. The oxidizing and stabilizer compound avoids aggregation at the moment nanoparticles are formed.
On the other side, the single-crystal cerium oxide nanoparticles of formula (I), in which n is higher than zero, thus the conjugated nanoparticles with n from 1 to 40, are obtainable by a method comprising the steps of:
(i) Preparing a solution 1 comprising albumin, polyvinylpyrrolidone, or mixtures thereof in a buffered solvent, particularly water, with a pH from 6.0 to 8.5;
(ii) Preparing a solution 2 of a cerium (III) salt in water wherein,
- if in the nanoparticle of formula (I) A is albumin, and optionally PVP, then an oxidizing and stabilizer compound is added in this solution 2, in particularly selected from TMAOH and HTM, and preferably TMAOH, and
- if in the nanoparticle of formula (I) A is only PVP next step (iii) is performed;
(iii) Adding solution 2 to solution 1 , wherein
- if in the nanoparticle of formula (I) A is only PVP, an oxidizing and stabilizer compound is added in this step (iii), in particularly selected from TMAOH and HTM, and preferably TMAOH; and
(iv) Let the mixture react for a period of time enough to obtain single- crystal CeO2NPs (CNPs), with a crystal size diameter measured by TEM from 3 to 24 nm, in particular from 3 to 15 nm and more in particular from 3 to 12, and even more in particular from 3 to 7 nm, said CNPs being conjugated with albumin, PVP or mixtures thereof, thus being single-crystal cerium oxide nanopartides of formula (I) with n from 1 to 40, and having a hydrodynamic diameter from 10 to 32 nm, in particular from 1 1 to 23 nm, the hydrodynamic diameter measured by DLS.
In a particular embodiment, TMAOH is preferred in any of the steps wherein it is used, since it is non-toxic to cells. In another particular embodiment, the cerium (III) salt is cerium (III) nitrate. TMAOH acts as oxidizing agent, as surfactant and also as stabilizer because it avoids nanoparticle aggregation.
In another preferred embodiment, the buffered solvent in (i) is phosphate- buffered saline (PBS) with a pH from 6.0 to 8.5 that comprises water and salts selected from sodium hydrogenphosphate, sodium chloride and mixtures thereof, and optionally potassium chloride and potassium
dihydrogenphoshate. In another particular embodiment of the method, from which single-crystal cerium oxide nanopartides in which n is from 1 to 40 are obtainable, solution 2 has a pH below 9, preferably from 6.0 to 8.5, more preferably from 7.0 to 8.0.
As a general rule, when the CeO2NPs are synthesized, the pH of the solution decreases and the surface charge of the nanoparticle decreases losing the ability to remain un-aggregated thanks to electrostatic repulsion. Procedures to de-aggregate CeO2NPs after synthesis have been proven unsuccessful (despite they work for other metal oxides as iron oxide). Therefore, the stabilizer for colloidal stability and to avoid CeO2NPs aggregation (in this particular case of the invention, the stabilizers being PVP, albumin or a combination of both) has to be administrated before aggregation takes place, which is immediately after nucleation, this is, at the beginning of the synthesis process. Besides, other stabilizers like TMAOH can be added during synthesis.
In another particular embodiment, the cerium (III) salt is particularly cerium (III) nitrate, and in yet another more particular embodiment it is in a concentration at solution 2 to give an initial cerium (III) concentration from 9.0 mM to 10.0 mM. In a particular embodiment of the single-crystal cerium oxide nanopartides obtainable by this method, the final concentration of albumin in solution 1 is from 0.70 to 0.80 mM, more particularly 0.75 mM.
In all these embodiments, during steps (iii) and (iv) exchange of adsorbed TMAOH by albumin, PVP or the combinations of both on the surface of cerium oxide nanopartides takes place. Residual traces of TMAOH can be present in the final single-crystal cerium oxide nanopartides of formula (I). When added, TMAOH is preferably added dropwise. Thus, in a particular embodiment, the single-crystal cerium oxide
nanopartides of formula (I) of the invention, wherein NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm; A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and n is an integer from 1 to 12, are obtainable by a method comprising the steps of:
(v) Preparing a solution 1 comprising albumin, polyvinylpyrrolidone, or mixtures thereof in a buffered solvent, particularly water, with a pH from 6.0 to 8.5;
(vi) Preparing a solution 2 of a cerium (III) salt in water wherein,
- if in the nanoparticle of formula (I) A is albumin, and optionally PVP, then an oxidizing and stabilizer compound is added in this solution 2, in particularly selected from TMAOH and HTM, and preferably TMAOH, and
- if in the nanoparticle of formula (I) A is only PVP next step (iii) is performed;
(vii) Adding solution 2 to solution 1 , wherein
- if in the nanoparticle of formula (I) A is only PVP, an oxidizing and stabilizer compound is added in this step (iii), in particularly selected from TMAOH and HTM, and preferably TMAOH; and
(viii) Let the mixture react for a period of time enough to obtain single- crystal CeO2NPs (CNPs), with a crystal size diameter measured by TEM from 3 to 7 nm, said CNPs being conjugated with albumin, PVP or mixtures thereof, thus being single-crystal cerium oxide nanopartides of formula (I) with n from 1 to 12, and having a hydrodynamic diameter from 1 1 to 23 nm, the hydrodynamic diameter measured by DLS.
With these methods, and as above reasoned, aggregation of the nanopartides does not take place, thus the nanoconjugates are constituted by single-crystal nanopartides, duly conjugated with albumin, PVP or both. In addition, these single-crystal cerium oxide nanopartides of formula (I), in which n is higher than zero, thus the conjugated nanopartides with n from 1 to 40, and obtainable as indicated in any of the above embodiments, are catalytically active single- crystal cerium oxide nanopartides. The nanopartides being catalytically active means that they retain its superoxide dismutase (SOD) and catalase mimetic activity. This catalytically active feature can be measured by many technologies, such as for example, by means of the CeO2NPs antioxidant effect on fluorescently-labeled hydrogen peroxidase (EuTc-H2O2 complex), Amplex Red or 2', 7' -dichlorofluorescin diacetate (DCFDA). Particular pharmaceutical or veterinary compositions comprising the nanopartides as defined above, are those further comprising physiologic saline buffer, such as Phosphate-buffered saline (PBS). By physiologic saline buffer is to be understood a water-based salt solution with osmolarity, pH (6- 7.5) and ion concentrations of mammal bodies, in particular of humans.
Particular salts and ions are sodium hydrogenphosphate, sodium chloride and optionally potassium chloride and potassium dihydrogenphoshate. In another particular embodiment, the pharmaceutical or veterinary compositions comprise the serum of the individual mammal to which the composition is to be administered.
Throughout the description and claims the word "comprise" and variations of the word, are not intended to exclude other technical features, additives, components, or steps. Furthermore, the word "comprise" encompasses the case of "consisting of. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention. Furthermore, the present invention covers all possible combinations of particular and preferred embodiments described herein.
EXAMPLES
Characterization techniques 1 ) Transmission microscopy:
Morphology of the engineered NPs was analyzed by High-Resolution
Transmission Electron Microscopy (HR-TEM, FEI Tecnai G2 F20 200 kV). The ultrafin 200-mesh copped TEM grids (Ted-Pella, Inc.) were placed on a filter paper. Afterwards, fifty microliters of the NPs colloidal suspension were deposited drop by drop over the grid and left to dry in air. The obtained HR- TEM images were analyzed by Image J program: for each sample, at least 150 NPs were measured and the average size and size-distribution were obtained.
2) UV-Vis Spectroscopy: UV-visible spectra was acqired with Schimadzu UV-2400 spectrofotometer. Regarding the detection limits of the apparatus and a high concentration of the synthesis, an aliquot of the synthesis solution was diluted in water (1/15) and than measured in a wavelength range between 260 and 800 nm.
3) ZP. DLS: A 1 ml aliquot of the synthesis solution was placed in a sizing couvette and analyzed by a Zetasizer Nano-ZS (Malvern Instruments). Thus, the surface charge (Zeta Potential, ZP), the Dynamic Light Scattering (DLS) and the Isoelectric Point (pi) of the nanoparticles were measured.
4) X-rav diffraction (XRD): A 10 ml aliquot of the synthesis solution was centrifuged twice (45 min at 12.000g), in order to ensure a maximal precipitation of the nanoparticles. A supernatant was discarded and the precipitates were dried at room
temperature for 2 days, to avoid further NP transformations. The data were colleted on a PANalytical X-Pert PRO MPD diffractometer using a Cu Ka 1 radiation source (λ = 1 .541 A). The 2Θ diffraction (Bragg) angles were measured by scanning the gliometer from 20° to 100°.
The peak positions and their width at half maximum (FWHM) were determined by the X-Pert High Score program after the baseline correction.
Example 1 . Synthesis of cerium oxide nanoparticles.
To obtain lipopolysaccharide (LPS)-free CeO2NPs, the synthesis was performed in fume hoods, replacing distilled water with pure milliQ water (18.2 ΜΩ conductivity and < 5 ppb TOC). Before the synthesis started, all the glass material and stirring bars were heat treated in an oven at 260°C for 1 h;
naturally cooled down to room temperature and rinsed with milliQ water (to remove calcined traces). The consistent use of gloves and sterile pipette tips was also strict.
1 .1 . Synthesis of PVP-coated 5nm CeO2 NPs:
An amount of 100ml of milli-Q water was placed under vigorous stirring (500 rpm) in a glass bottle at room temperature. Then, 5g of Polyvinylpyrrolidone (PVP of 10kDa, Sigma Aldrich) was added, to give a 5mM concentration of
PVP. The solution was left at room temperature under vigorous stirring (500 rpm) for 30 min, until all solid was homogeneously dissolved. Besides, a solution of cerium (III) nitrate hexahydrated was prepared in water with 0.434 g of cerium (III) nitrate hexahydrated (Ce(NO3)3-6H2O, 99%, Sigma). This solution was added to that of PVP, to give a 10 mM concentration of initial cerium (III) precursor. The solution was vigorously mixed during 15 minutes. Just after that, 2.2 ml of an as-received TMAOH (1 M solution) was gently added dropwise during half of a minute (20-30 seconds), approximately. The solution suffered several changes in color, from pale yellow to whitish, during the first two hours of reaction. 24h later, the PVP-coated 5nm nanoparticles were ready at 1 mg/ml concentration and stored at 4°C.
1 .2 Synthesis of BSA-coated 5 nm CeO2 NPs:
Solution 1 . An amount of 25 ml of PBS (Dulbecco 's phosphate buffered saline 10x, Sigma) was placed under vigorous stirring (500 rpm) in a glass bottle at room temperature. Then, 0.625 g of bovine serum albumin (BSA, Sigma) was added under vigorous stirring, to give a homogeneous solution at 0.752 mM concentration.
Solution 2. In parallel, in another glass bottle, an amount of 25 ml of milli-Q water was placed under vigorous stirring (500 rpm). Then, 0.217 g of cerium (III) nitrate hexahydrate (Ce(NO3)3-6H2O, 99%, Sigma) were added, to give a 10 mM concentration of initial cerium (III) precursor. Following, a volume of 1 .1 ml of an as-received TMAOH (Sigma, 1 M) was gently added dropwise, during half of a minute approximately (dropwise for a period from 20 to 30 seconds). The reaction was left under vigorous stirring during five minutes. After this time, the solution 2 was gently added to the solution 1 under vigorous stirring during one minute approximately. The resultant solution was left at room temperature and under vigorous stirring for 24 h. 24 h hours later, the BSA-coated 5nm CeO2NPs were ready and stored at 4°C. 1J3. Monitoring of CeO2NPs formation with UV-vis spectroscopy:
The optical study with ultraviolet-visible spectroscopy (UV-vis) was carried out to corroborate the NPs formation (any of 1 .1 ; PVP-conjugated, or 1 .2; BSA- conjugated) and to monitor the reaction kinetics. Despite the unspecificity of UV light absorption at short spectra wavelengths, it resulted in a good technique to monitor the progress and evolution of the reaction. FIG. 3 (A) shows pH evolution of the synthesis solution, meanwhile FIG. 3 (B)
corresponds to the non-oxidized initial Cerium (III) precursor (black line); of the sample taken at 1 h of reaction (soft grey line) and of the sample of as- synthesized NPs at 48 h of reaction. A strong characteristic UV absorption peak of Ce4+ at 298 nm, confirms particles formation (H.L.Greenhaus et al., Ultraviolet Spectrophotometnc Determination of Cerium(lll), Anal Chem -1957, vol. no. 29, pp.: 1531 ).
1 .4. Nanoceria characterization by Electron Microscopy: To minutely examine the quality of produced CeO2NPs, in terms of
morphology, size, aggregation state and chemical composition (purity), the samples were analyzed by high resolution electron microscopy (HR-TEM, STEM-HAADF, EELS). A few representative images of as-synthesized PVP- coated samples are displayed in FIG. 4. Corresponding (similar) images were recorded with BSA-coated NPs. FIG. 4 (a) shows as-synthesized
monodispersed PVP-coated NPs at high resolution (400.000X), displaying a particle size of 5.2 ± 1 .9 nm (Image J analysis) and the atomic planes of single-crystal nanoparticles. FIG. 4 (b) shows the dark-field STEM-HAADF image of the NPs, while FIG. 4 (c) inspects the homogeneity and
monodispersity degree of nanoceria at lower magnification (70.000X). FIG. 4 (d) shows a typical single-crystalline CeO2 NP and its correspondent Fourier Transformation, FFT (FIG. 4 (e)). The profile of the chemical composition, represented in FIG. 4 (f) displays absolute purity of the sample. 1J5. Nanoceria characterization by X-ray diffraction:
The structural and compositional characterization, phase identification and size of CeO2NPs were analyzed by X-ray Diffraction (XRD). The XRD pattern of as-synthesized CeO2NPs of 4 nm is displayed in FIG. 5. The XRD pattern was scanned from 20 to 80 degrees and the XRD profile confirmed the monocrystalline nature of CeO2NPs (single-crystal cerium oxide
nanoparticles). The characteristic diffraction peaks were observed at 2Θ = 28.51 °, 33.12°, 47.44°, 56.30°, 58.88°, 69.36°, 76.75°, 79.02°, 88.35° and 95.34° correlated to (1 1 1 ), (200), (220), (31 1 ), (222), (400), (331 ), (402), (422), (333) crystal planes, respectively. All of the reflection peaks are in agreement with the database standard (JCPDS 34-0394) of the face-centered cubic CeO2 crystal with the fluorite structure.
1 .6. Crystal size diameter estimation (Crystal size estimation) Three techniques were used to estimate the average crystal size diameter of as-synthesized in 1 .1 or 1 .2 CeO2NPs: Image J analysis software for HR- TEM; size estimation using the Scherrer equation of XRD pattern and the Dynamic Light Scattering (DLS) of colloidal suspension of the NPs (measuring hydrodynamic diameter). Table 1 shows a comparison between three sizing techniques for as-synthesized CeO2NPs: (a) Dynamic Light Scattering (DLS) and the polydispersity index (PDI) of this DLS, (b) Image J analysis of HR- TEM images, and (c) Scherrer equation calculations from XRD profile. All the data suggests good agreement for all methods regarding the NPs size. PDI is a parameter in DLS related with size distribution of molecules or particles in suspension. For a perfectly uniform sample, the PDI would be 0.0. PDI from 0.0 to 0.1 reflects a narrow non-uniform distribution type sample. From 0.1 to 0.4 the PDI indicates a moderate non-uniform distribution type sample. PDI higher than 0.4 is usually understood as a broad distribution type sample.
Table 1
Figure imgf000025_0001
The size (diameter in nm) and aggregation state of the CeO2NPs conjugated with albumin disclosed in 1 .2 were again corroborated by DLS technique. In both measurements, by intensity and by number, it was observed one single peak at around 12 nm (hydrodynamic diameter). The absence of peaks at larger size axis corroborates the absence of aggregates in the designed synthesis method. Moreover, as an internal control, the BSA-coated NPs were purified by centrifugation and measured by DLS the supernatant only (FIG. 6 (c) and (d)), that contains free BSA in excess. The size distribution coincides with previously obtained data in FIG. 6 (a) and (b), wherein the DLS of the as- synthesized BSA-coated CeO2NPs is depicted. All these data allow concluding that single-crystal cerium oxide nanoparticles of formula (I) (NP-(A)n) were conjugates in which NP had a crystal diameter from 3 to 7 nm, around 4-5 nm, when measured with TEM and other techniques, such as XRD. In addition albumin or PVP was disposed conjugated (as a coating) with n from 1 to 4, particularly n was 1 . When n is greater, the single peak appears in a position with higher nm in a graphic as that of FIG. 6.
Example 2. In vivo assay
An in vivo assay was performed with 44 Wistar rats to which HCC was induced by means of diethylnitrosamine (DEN). DEN is an hepatic
carcinogenic used commonly to induce HCC in animals. All animals received weekly an injection intraperitoneally (50 mg of DEN/Kg) for 16 weeks. Two protocols were carried out:
In first protocol the effect of CeO2 NPs of Example 1 .2 (BSA-conjugated CNPs) on hepatic morphology, conventional serology and cell proliferation were studied. In weeks 16 (last of DEN injection) and 17, rats were randomly divided into two groups of 10 animals per group, each group receiving, respectively, two doses of CeO2 NPs (0.1 mg/Kg) administered intravenously weekly for two weeks (Test group). The other group (Control) received the vehicle (TMAOH, 0.8 mM). At week 18, rats were sacrificed and blood and liver samples were obtained to study standard hepatic functional parameters and hepatocyte proliferation.
In the second protocol (24 rats), the effect of BSA-conjugated CNPs of Example 1 .2 on survival of animals with induced HCC was studied. The same procedure as in the first protocol was followed with the difference that animals were sacrificed only when a badly damaged state was evident and always following criteria of Ethical and Research Committee of Hospital Clinic (Barcelona).
Liver sections of non-treated with CeO2 NPs rats had a more dysmorphic appearance also with more superficial deformities than liver of CeO2 NPs treated rats. In the same way, the liver weight percentage in relation to total body weight was slightly lower in animals receiving CeO2 NPs in comparison with non-treated animals. Non meaningful differences were observed in serologic standard hepatic functional parameters and in renal parameters between bot animal groups.
The most important result of the first protocol was that hepatic cell proliferation measured as the percentage of hepatocyte nuclei positively stained (tinted) with Ki-67 was markedly lower in hepatic tissue of CeO2 NPs treated rats than in the non-treated rats (21 ±2 vs 52±5, p<0.001 ). Data are depicted in FIG. 1 (A and B), wherein the microscope image (FIG. 1 (A)) of the liver of non- treated cells (VEHICLE) shows a higher percentage of Ki-67 stained
hepatocyte nuclei than in the image of CeO2 NPs treated liver (CeO2NPs). Same results can be concluded from FIG. 1 (B), wherein for all animals of the protocol the percentage of stained Ki-67 nuclei are depicted. Lower
percentages are observed in treated animals, thus demonstrating that the treatment with the CeO2 NPs reduced cell proliferation.
In the second protocol 24 rats were randomly treated with CeO2 NPs (n=12) or with vehicle (n=12). This assay showed that survival of HCC rats treated with CeO2 NPs was higher than in non-treated rats. At week 21 , 4 weeks after last dose of CeO2 NPs or of vehicle, percentage of survival of non-treated rats was lower than in treated rats (10 %). This value increased up to 40 % in treated rats. Data are depicted in FIG. 2. All these data demonstrated that CeO2 NPs have a powerful effect on cell proliferation and on rat survival with HCC, thus being useful in the treatment of HCC patients.
Example 3. CeO2 NPs for use in combination with radiotherapy.
FIG. 7 is an image of X ray contrast imaging of samples at different CeO2NPs concentrations ranging from 0 to 100 mg/ml . The samples where imaged at 90 kV and 160 microA. The bright white signal, indicative of X-ray absorption increases with CeO2 concentration as expected. This X-ray absorption is at the basis of X-ray imaging and X-ray therapy.
As shown in this FIG. 7, CeO2 NPs, absorbed X radiation, in a highly efficient manner. Transfer of energy was also high with these nanoparticles. Therefore, they are good contrast agents (in particular as X-ray contrast agents), as well as good to be used in combination with radiotherapy, providing to the subject the intrinsically effect of the ceria nanoparticles (due to their superoxide dismutase (SOD) and catalase mimetic activity), and enhancing the effect of the administered radiotherapy.
Example 4. Catalytic activity of PVP-conjugated CeO2NPs (single-crystal cerium oxide nanoparticles of formula (I) in which A is PVP)
Catalytic activity of PVP-conjugated CeO2NPs was studied by detection of the the CeO2NPs antioxidant effect on fluorescently-labeled hydrogen peroxidase (EuTc-H2O2 complex). There were used:
(a) Commercial 25-nm-sized CeO2NPs (aggregates)
(b) PVP-coated 5 nm-sized CeO2NPs of the invention (synthesized as in Example 1 );
(c) surfactant-free mix of CeO2NPs (mix of sizes from 6 to dozens of nm), which where thus naked (not conjugated) CeO2NPs;
(d) surfactant-free 5-nm-sized CeO2NPs, which where thus naked (not conjugated) CeO2NPs.
Results are depicted in FIG. 8, a graphic displaying for each assayed nanoparticle type the decay of fluorescence (F) versus time (t, in min).
In cell-free environment, as bigger the diameter of CeO2NPs, lower their antioxidant capacity, which is displayed by an inefficient decay of fluorescence (F) versus time (t).
The PVP-coating slightly decreases CeO2NPs reactivity, but as deducible from FIG. 8, PVP-coated 5nm-sized CeO2NPs of the invention maintained the catalytic activity, and not so did commercial.
REFERENCES CITED IN THE APPLICATION
- ANN et al., "Downregulation of Tumor Growth and Invasion by Redox- Active Nanoparticles", Antioxidant & Redox Signaling (Original Research Communication) - 2013, vol. 00, pp.: 1 -14 (DOI:
10.1089/ars.2012.4831 ).
Chen et al., in "Cerium oxide nanoparticles induce cytotoxicity in human hepatoma SMMC-7721 cells via oxidative stress and the activation of MAPK signalling pathways", Toxicology in vitro - 2013, vol. no. 27, pp.: 1082-1088.
Hak Soo et al., "Renal clearance of quantum dots", Nature
biotechnology - 2007. vol. no. 25(10), pp.: 1 165-1 170.
Bushan et al., "Antioxidant nanozyme: a facile synthesis and evaluation of the reactive oxygen species scavenging potential of nanoceria encapsulated albumin nanoparticles", Journal of Materials Chemistry B - 2015, vol. no. 3, pp.: 4843-4852.
Marsalek et al., "Adsoprtion of Bovine Serum Albumin on CeO2",
International Journal of Chemical. Nuclear, Materials and Metallurgical Engineering -2014, Vol. no. 8(12), pp.: 1269-1272.
H.L.Greenhaus et al., Ultraviolet Spectrophotometric Determination of Cerium(lll), Anal Chem -1957, vol. no. 29, pp.: 1531 .

Claims

1 . - Single-crystal cerium oxide nanoparticle of formula (I) NP-(A)n (I), wherein
NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 24 nm;
A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and
n is an integer from 0 to 40, for use in the treatment of hepatocellular carcinoma, and wherein if the crystal diameter of the cerium oxide nanoparticle is from 3 to 7 nm, then n is an integer from 1 to 12.
2. - The single-crystal cerium oxide nanoparticle for use according to claim 1 , wherein NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 24 nm; A is a molecule selected from the group consisting of albumin,
polyvinylpyrrolidone, and combinations thereof; and
n is an integer from 1 to 40.
3. - The single-crystal cerium oxide nanoparticle for use according to any one of claims 1 -2, in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 12 nm.
4. - The single-crystal cerium oxide nanoparticle for use according to any one of claims 1 -3, in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm, A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and
n is an integer from 1 to 12.
5. - The single-crystal cerium oxide nanoparticle for use according to claim 4, wherein NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm.
6. - The single-crystal cerium oxide nanoparticle for use according to any one of claims 1 -4, in which NP is a cerium oxide nanoparticle with a diameter from 3 to 7 nm, A is a molecule of albumin; and n is an integer from 1 to 12.
7. - The single-crystal cerium oxide nanoparticle for use according to claim 6, wherein NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm, and n is from 1 to 4.
8. - The single-crystal cerium oxide nanoparticle for use according to any one of claims 1 -4, in which NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm, A is a molecule of polyvinylpyrrolidone; and n is an integer from 1 to 12.
9. - The single-crystal cerium oxide nanoparticle of formula (I) for use according to any one of claims 1 -8, which is for use as inhibitor of cell proliferation in hepatocellular carcinoma.
10. - The single-crystal cerium oxide nanoparticle of formula (I) for use according to any one of claims 1 -9, which is for use as a co-treatment of an hepatocellular carcinoma therapy selected from the group consisting of radiotherapy, microwave ablation (MWA), radiofrequency ablation (RFA), transarterial radioembolization (TARE), chemotherapy, surgical resection, liver transplantation, targeted therapy, hyperthermia and combinations thereof.
1 1 . - A single-crystal cerium oxide nanoparticle of formula (I)
NP-(A)n (I), wherein
NP is a cerium oxide nanoparticle with a crystal diameter from 3 to 7 nm;
A is a molecule selected from the group consisting of albumin, polyvinylpyrrolidone, and combinations thereof; and
n is an integer from 1 to 12.
12. - The single-crystal cerium oxide nanoparticle according to claim 1 1 , wherein NP is a cerium oxide nanoparticle with a crystal diameter from 4 to 5 nm.
13.- The single-crystal cerium oxide nanopartide according to any one of the claims 1 1 -12, wherein A is a molecule of albumin; an n is an integer from 1 to 4.
14.- A single-crystal cerium oxide nanopartide of formula (I): NP-(A)n (I), wherein
NP is a cerium oxide nanopartide with a crystal diameter from 8 to 24 nm;
A is a molecule of albumin; and
n is an integer from 5 to 40.
15. - A pharmaceutical or veterinary composition comprising single-crystal cerium oxide nanopartide as defined in any of claims 1 1 -14, together with one or more pharmaceutically or veterinary acceptable excipients or carriers.
16. - The pharmaceutical or veterinary composition according to claim 15 comprising physiologic saline buffer.
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