Hybrid ceria and gold nanoparticles for use in simultaneous medical diagnosis and radio-protection and theragnostic agent comprising them

Abstract

Au@CeO2 nanoparticles comprising an Au core having an average diameter from 5 to 100 nm and a CeO2 shell having a thickness from 5 to 20 nm for use in simultaneous imaging diagnosis and radio-protection are disclosed. It is also disclosed a theragnostic agent comprising Au@CeO2 nanoparticles and pharmaceutically acceptable excipients and a process for its preparation; wherein the theragnostic agent is in the form of an colloidal solution, and wherein the Au@CeO2 nanoparticles are at a concentration from 10 to 100 mg / mL; wherein the theragnostic agent is a dual-purpose radio-protecting and contrast agent. A process for the preparation of the theragnostic agent.

Description

[0001] Hybrid ceria and gold nanoparticles for use in simultaneous medical diagnosis and radio-protection and theragnostic agent comprising them

[0002] This application claims the benefit of European Patent Application EP24382664.1 filed on June 19th2024.

[0003] Technical Field

[0004] The present invention relates to hybrid Au@CeC>2 nanoparticles for use in simultaneous medical diagnosis and radio-protection, to a theragnostic agent comprising the Au@CeC>2 nanoparticles, and to a process for preparing the mentioned agent.

[0005] Background Art

[0006] X-ray computed tomography (CT) stands as a powerful diagnostic tool capable of delivering deep tissue penetration and 3D anatomical imaging of the entire body with high resolution. To enhance image contrast, patients routinely receive contrast agents, typically composed of elements with high atomic numbers (high-Z). Currently, iodine-based molecules are the most employed contrast agent for medical imaging due to their low molecular weight and high solubility, and therefore good in vivo dispersibility. However, these agents have significant drawbacks, primarily related to their poor pharmacokinetics. They exhibit extremely short circulation times in the bloodstream and often are cleared from the body within seconds to minutes, limiting their utility. Consequently, patients frequently are administered large doses of these molecules to attain the desired image contrast enhancement, potentially leading to dose-related toxic side effects such as nephropathy.

[0007] Moreover, the increased use of CT scans has raised concerns about risk of exposure to ionizing radiation. Recent studies have clearly observed an elevation in DNA damage and alterations in gene expression following CT scans. In fact, many countries have begun to limit the number of CT scans performed on children.

[0008] Hence, there is a growing demand for safer alternative contrast agents. Small nanoparticles composed of heavy atoms have garnered significant attention for some potential advantages over commercial iodine-based compounds. They possess higher X- ray attenuation coefficients, allowing for more efficient X-ray absorption, and they present longer circulation times, extending the imaging window. This property enables targeted imaging and reduces the required administration volumes. Additionally, NPs exhibit heterogeneous distribution in organs depending on tissue porosity, which is linked to tissue health status. Accordingly, various high-Z elements have been explored as viable substitutes. There have been several recent studies where nanoparticles whose payload is gold, bismuth, tantalum, or other elements have been proposed as contrast agents for CT. However, the use of contrast agents during CT scans further exacerbates the radiation dose delivered to organs where the contrast agent is, resulting in an increased occurrence of double-strand DNA breaks, which are the most challenging to repair.

[0009] Garcia A. et al. discloses the potential of cerium oxide NPs conjugated with murine serum albumin (CeChNPs-MSA) as a contrast agent for X-ray CT imaging, primarily due to their high X-ray attenuation, excellent biocompatibility, and radioprotective properties. Nevertheless, it is pointed out that further research and development in this area is needed for the translation of CeChNPs-MSA into clinical applications for enhanced medical imaging and improved diagnosis of diseases.

[0010] Jain V, et al. discloses mercaptoundecanoic acid (11-MUA) coated Au core and CeC>2 shell nanoparticles (CSNPs) and their enzyme-like activity. However, a loss of superoxide dismutase (SOD) and catalase enzyme-like activities was observed, compromising the multifunctional property of CSNPs.

[0011] CN118001394 discloses an Au@CeO2' polypeptide nanocomposite material useful for the treatment of osteoarthritis.

[0012] Zhang W et al. discloses Au@CeO2 nanoparticles in solution at a dosage of 1.5 mg / kg for NIR facilitating purposes and to reduce inflammation.

[0013] Thus, there is still a need in the field of medical imaging of new contrast agents for X-ray imaging and CT and, particularly, for theragnostic.

[0014] Summary of Invention

[0015] Inventors have found by using Au@CeC>2NPs as contrast agent in imaging diagnosis, an unexpected efficient image contrast at various incident tube potentials comparable to clinical iodinated agents is obtained.

[0016] Surprisingly, inventors realized that by incorporating an Au core in ceria-based nanoparticles, X-ray contrast (e.g., p / pAu = 5.2 cm2 / g at 100 keV) was enhanced while protecting against reactive oxygen species (ROS) species thanks to the CeO2 shell. As shown in the examples, the Au@CeO2NPs presented an increased antioxidant capability, achieving a remarkable ROS reduction of the H2O2-induced ROS production by 75% for Au@CeO2NPs compared with the by 60% for the same mass of CeO2NPs, as can be seen in Fig. 4B. Thus, Au@CeO2NPs disclosed herein provides an unexpectedly good ROS reduction, thus providing radioprotection when used as contrast agent in imaging diagnosis. Also as shown in the examples, notably, Au@CeC>2NPs of the present disclosure proved highly effective at enhancing X-ray CT image contrast under both lower and higher tube potentials (< 70 kVp and > 90 kVp). Conversely, CeChNPs, not having a core of gold, offered optimal contrast only at intermediate tube potentials (70-90 kVp).

[0017] Hence, Au@CeC>2NPs of the present disclosure are particularly efficient for clinical X-ray computed tomography, mammography, chest imaging, abdominal imaging, and paediatric imaging, among other applications.

[0018] Advantageously, the combined unexpectedly good imaging at the above mentioned tube potentials and radio-protection capacities of Au@CeC>2NPs disclosed herein (due to their ability to provide protection against ROS) allows for simultaneous imaging for diagnosis while treating the body from diagnosis damage, combining both aspects into theragnostics. This achievement stands as a significant leap forward not only compared to traditional contrast agents but also comparted to CeO2NPs, since ROS generation is the principal culprit behind radiation-induced damage and the associated dose-limiting effects.

[0019] X-ray experiments with phantoms have shown that current Au@CeO2NPs provide an efficient contrast enhancement compared with iodinated agents, and also compared to CeO2NPs at some tube potentials.

[0020] As mentioned above, whereas CeO2NPs optimize the X-ray attenuation at intermediate tube potentials (70-90kVp), Au@CeO2NPs improve the contrast signal at lower and higher tube potentials, while having exceptional ROS scavenging properties.

[0021] This constitutes a noteworthy improvement compared not only to iodine-commercial products and other high-Z nanoparticles, such as AuNPs, but also compared to CeO2NPs, because ROS products generated during irradiation are the main responsible for radiation-induced damage.

[0022] Thus, a first aspect of the present disclosure relates to Au@CeO2 nanoparticles consisting of an Au core having an average diameter from 5 to 100 nm and a CeO2 shell having an thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeO2 shell completely covers the surface of the Au core, for use in simultaneous imaging diagnosis and radio-protection.

[0023] A second aspect of the present disclosure relates to a theragnostic agent comprising i) Au@CeC>2 nanoparticles consisting of an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core; and ii) pharmaceutically acceptable excipients; wherein the theragnostic agent is in the form of an colloidal solution, and wherein the Au@CeC>2 nanoparticles are at a concentration from 10 to 100 mg / mL; wherein the theragnostic agent is a dual-purpose radio-protecting and contrast agent.

[0024] A further aspect relates to a theragnostic agent wherein the theragnostic agent is for use in simultaneous imaging diagnosis and radio-protection, the theragnostic agent comprising the Au@CeC>2 nanoparticles as defined above and pharmaceutically acceptable excipients; wherein the theragnostic agent is in the form of an colloidal solution; and wherein the Au@CeC>2 nanoparticles are at a concentration from 10 to 100 mg / mL; wherein the theragnostic agent is a dual-purpose radio-protecting and contrast agent.

[0025] Therefore, this aspect relates to the use of the Au@CeC>2 nanoparticles defined herein for the manufacture of a theragnostic agent for simultaneous medical diagnosis and radioprotection, the theragnostic agent comprising the Au@CeC>2 nanoparticles as defined above and pharmaceutically acceptable excipients; wherein the theragnostic agent is in the form of an colloidal solution; and wherein the Au@CeC>2 nanoparticles are at a concentration from 10 to 100 mg / mL. Alternatively, this aspect may also be formulated as a method for medical diagnosis and rad io- protection in a mammal, including a human, the method comprising intravenously or intradermally administering to the mammal an effective amount of the theragnostic agent as defined herein.

[0026] Another aspect of the present disclosure refers to a process for the preparation of a theragnostic agent comprising the Au@CeC>2 nanoparticles as defined herein, and pharmaceutically acceptable excipients, the process comprising (a) preparing an initial colloidal solution of the Au@CeC>2NPs comprising an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core; (b) concentrating the initial colloidal solution obtained in step (a) until obtaining a concentrated colloidal solution comprising from 10 to 100 mg / mL Au@CeC>2 nanoparticles; and (c) adding to the concentrated colloidal solution the pharmaceutically acceptable excipients for intravenous or intradermal administration.

[0027] In another aspect, the present disclosure refers to the use of the Au@CeC>2 nanoparticles defined herein for the manufacture of an article of manufacture providing protection against ionizing radiation.

[0028] In another aspect, the present disclosure refers to an article of manufacture comprising the Au@CeC>2 nanoparticles disclosed herein.

[0029] Brief Description of Drawings

[0030] Fig. 1 shows: A-C) X-ray Attenuation by CeChNPs, Au@CeC>2NPs, and AuNPs at concentrations from 0 to 200 mM, measured at 50, 70, and 90 kVp; and X-ray attenuation by precursor salts: Ce(NC>3)3, HAuCk, and a mixture of Ce(NOs)3 + HAuCk for comparison. D-F) X-ray attenuation by CeChNPs and Au@CeC>2NPs, compared to AuNPs and clinical iodinate product lohexol at equimolar concentrations, at 50 kVp, 70 kVp, and 90 kVp.

[0031] Fig 2 shows: H-J) original CT images at maximum NP concentrations (200 mM) and their dilutions at X-ray tube voltages 50 kVp, 70 kVp, and 90 kVp. K) Conceptual representation of efficient X-ray attenuation with symbols of chemical elements (Ce for CeChNPs and Ce- Au for Au@CeC>2NPs) engraved into a 3D-printed plastic sheet, producing an X-ray image (70 kVp tube potential, 200 pA intensity).

[0032] Fig. 3 shows X-ray absorption images produced by the precursor salts (i.e., Ce(NC>3)3 , HAuCh, Ce(NC>3)3 + HAuCh mix, and lohexol product) at the maximum concentration (200 mM) and their dilutions.

[0033] Figure 4 shows the ROS scavenging capacity and cell viability of ceria-based nanoparticles and controls. (A) Assessment of H2O2 scavenging capacity using the Amplex Red test, with H2O2 concentrations ranging from 0-6 pM and measurement of the final H2O2 concentration after the reaction. (B) The scavenging capacity of CeO2NPs, Au@CeO2NPs, and AuNPs. (C) Comparison of H2O2 scavenging capacity using the Amplex Red test between uncoated CeO2 NPs and Au NPs and their conjugated counterparts employed in the CT experiments. (D) Comparison of H2O2 scavenging capacity of a second set of synthesized Au@CeC>2NPs. (E-F) Dose-response cytotoxicity assessment of CeChNPs and CeCh-AuNPs on MCF-7 and HeLa cells using the PrestoBlue assay, with NPs incubated for 24 hours at concentrations ranging from 1-100 pg / ml.

[0034] Fig. 5. H2O2degradation activity measured using the Amplex Red assay. The reduction of H2O2was monitored by quantifying the resorufin fluorescence signal (excitation / emission: 530 / 590 nm) produced in the presence of horseradish peroxidase and Amplex Red. a: Au@CeC>2NPs of Example 1 ; b: 11-MUA coated Au@CeC>2 NPs (Comparative Example 5); and c: the Au@CeC>2-PEG NPs (Comparative Example 6). All nanoparticle suspensions were tested at a concentration of 75 pM of elemental cerium to ensure comparability of catalytic activity

[0035] Detailed description of the invention

[0036] All terms as used herein in this application, 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 throughout the description and claims.

[0037] The term “nanoparticle or NP”, as used herein, refers to a particle with at least two dimensions at the nanoscale, particularly with all three dimensions at the nanoscale. As used herein "nanoscale" refers to dimensions from 1 to 150 nm and having any size, shape, or morphology. As regards the shape of the nanoparticles described herein, particularly, they include spherical and polyhedral nanoparticles. In a particular embodiment the nanoparticle is spherical or polyhedral, particularly spherical.

[0038] As used herein, the term "size" refers to the diameter, irrespective of the actual particle shape. For example, in the case of a nanoparticle that is substantially spherical, the size of the nanoparticle corresponds to the diameter of the nanoparticle. The term "diameter", as used herein, means the equivalent sphere diameter, namely the diameter of a sphere having the same diffraction pattern, when measured by laser diffraction, as the particle. When referring to a set of nanoparticles as being of a particular size, it is contemplated that the set of nanoparticles can have a distribution of sizes around the specified size. Thus, as used herein, a size of a set of nanoparticles 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.

[0039] The nanoparticle size may be measured using methods well known to those of skill in the art, such as by Transmission Electron Microscopy (TEM), and Dynamic Light Scattering (DLS).

[0040] The term Au@CeC>2 nanoparticles (Au@CeC>2NPs), as used herein, refers to Au / CeCh hybrid nanoparticles comprising a Au core coated with a CeC>2 shell.

[0041] The term "theragnostic", as used herein, being derived from the Greek words therapia and diagnosis, refers to a combination of diagnostic and therapy. Particularly, in the context of the present disclosure, the term "theragnostic agent" refers to a dual-purpose radioprotecting agent and contrast agent. In the context of the present disclosure, therapia refers to a prophylactic treatment.

[0042] As used herein, the expression "effective amount" with respect to a therapeutic or diagnostic agent refers to the amount of the therapeutic / diagnostic agent that, when administered to a human or veterinary patient, is sufficient to exert the intended diagnostic or therapeutic effect. The particular dose of therapeutic / diagnostic agent administered according to this invention will of course be determined by the particular circumstances surrounding the case, including, the route of administration, the particular condition being diagnosed / treated, the particular circumstances of the individual subject to be diagnosed / treated, and similar considerations. The term "colloidal solution" or "colloid" refers to a mixture comprising particles of one component (in the context of the present disclosure, Au@CeC>2 nanoparticles) that are evenly distributed (but not dissolved) within a medium in such a way that they can not settle nor separate from the medium over time.

[0043] The term 'dual-purpose', as used herein, mean that the contrast agent of the present invention is simultaneously providing radio-protection, and thus is providing both medical diagnosis and prophylactic treatment by means of radio-protection.

[0044] As used herein, the indefinite articles “a” and “an” are synonymous with “at least one” or “one or more.” Unless indicated otherwise, definite articles used herein, such as “the” also include the plural of the noun.

[0045] As mentioned above, a first aspect of the present invention relates to Au@CeC>2 nanoparticles consisting of an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell have a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core, for use in simultaneous imaging diagnosis and radio-protection.

[0046] In an embodiment, the CeC>2 shell has a thickness from 5 to 10 nm.

[0047] In another embodiment, the CeC>2 shell has a thickness from 5 to 5.5 nm.

[0048] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the Au core has an average diameter from 5 to 15 nm, particularly of 9-10 nm.

[0049] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the CeC>2 shell comprises CeC>2 crystals of 2-3 nm in size.

[0050] In another embodiment, the Au@CeC>2 nanoparticles for use of the present disclosure have a size from 10 to 60 nm, particularly from 5 to 20 nm, and more particularly from 10 to 15 nm, determined by TEM.

[0051] As mentioned above, another aspect of the invention relates to a theragnostic agent as defined above.

[0052] In an embodiment of the theragnostic agent, the CeC>2 shell has a thickness from 5 to 10 nm.

[0053] In another embodiment of the theragnostic agent, the CeC>2 shell has a thickness from 5 to 5.5 nm. In another embodiment of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the Au core has an average diameter from 5 to 15 nm, particularly of 9-10 nm.

[0054] In another embodiment of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, wherein the CeC>2 shell comprises CeC>2 crystals of 2-3 nm in size.

[0055] In another embodiment of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the nanoparticles have a size from 10 to 60 nm determined by TEM.

[0056] In another embodiment of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the nanoparticles have a size from 10 to 15 nm determined by TEM.

[0057] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the pharmaceutically acceptable excipients comprise water for injection and sodium citrate or citric acid.

[0058] Advantageously, the sodium citrate present in the colloidal solution imparts stability through electrostatic repulsion to the Au@CeC>2 nanoparticles. Particularly, by stabilizing the surface of as-synthesized Au@CeC>2NPs, a high mass concentrations can be reached with good colloidal stability, what is advantageous in clinical CT scans. Au@CeC>2NPs also show promising biological compatibility as they remain stable in physiological media at 37 °C without aggregation and do not induce any significant cytotoxicity at concentrations up to 100 pg / mL (see Fig. 4E-F).

[0059] In another embodiment of the theragnostic agent of the present disclosure, optionally in combination with one or more features of the various embodiments described above, the Au@CeC>2 nanoparticles are at concentration from 15 to 50 mg / mL, more particularly from 20 to 50 mg / mL or from 20 to 40 mg / mL.

[0060] Given the substantial concentrations of contrast agents required in clinical CT imaging, the inventors assessed the colloidal stability of NP samples at high concentrations (e.g., >100 mM / ; >20 mg / mL). In these conditions, as shown in the examples, the results demonstrate that the synthesized NPs remained colloidally stable under physiological conditions without affecting cell viability.

[0061] As mentioned above, the combined unexpectedly good imaging and radio-protection capacities of Au@CeC>2NPs allow for simultaneous enhanced medical imaging for improved diagnosis of diseases while treating the body from diagnosis damage. Thus, another aspect relates to a theragnostic agent as defined above, wherein the theragnostic agent is for use in simultaneous imaging diagnosis and radio-protection, particularly, wherein the pharmaceutically acceptable excipients comprise water for injection and sodium citrate or citric acid.

[0062] While CeC>2 NPs enhance X-ray absorption at intermediate tube potentials (70-90 kVp), medical imaging requires adjusting X-ray energy levels for specific anatomical regions. Chest imaging requires 100-140 kVp, whereas abdominal imaging uses 80-120 kVp. Paediatric imaging minimizes radiation with lower energy (70-100 kVp), while mammography employs around 25-35 kVp. Whole-body imaging strategically combines energy levels to visualize diverse regions in a single scan. Enhancing contrast not only in the intermediate range but also at lower and higher energies ensures adaptability to varied clinical scenarios, providing a versatile solution for diagnostic imaging.

[0063] Therefore, in another embodiment, the theragnostic agent for use of the present disclosure, the simultaneous imaging diagnosis and radio-protection is used in radioguided microsurgery applications.

[0064] In another embodiment, medical diagnosis is performed by X-ray imaging, particularly, in dentistry.

[0065] In another embodiment, the theragnostic agent of the present disclosure is for use to provide radiopaque contrast in medical diagnosis, wherein medical diagnosis is performed by X-ray imaging, particularly for use as a substitute of tantalum in medical diagnosis.

[0066] In an embodiment, X-ray imaging is selected from the group consisting of contrast- enhanced mammography, urography, arteriography, phlebography, cardio angiography, cervical myelography, arthrography, hysterosalpingography, sialography, fluoroscopy, chest imaging, abdominal imaging, and paediatric imaging. Particularly, X-ray imaging is performed by X-ray computed tomography.

[0067] Particularly, medical diagnosis by X-ray imaging comprises visualization of abnormal structures or lesions and differentiation between healthy and pathological tissue in a mammal, including a human.

[0068] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the administration of the theragnostic agent of the present disclosure is intravenous or intradermal. In case of intravenous administration, it can be administered by injection or by infusion.

[0069] Generally, suitable formulations include aqueous and non-aqueous, isotonic sterile solutions having a suitable pH and stability, which can contain, for instance, anti-oxidant agents, buffers, bacteriostatic agents, suspending agents, solubilizers, thickening agents, stabilizers, and preservatives, among other pharmaceutically acceptable excipients.

[0070] A suitable administration amount of the theragnostic agent of the present disclosure may vary depending on the particular circumstances of the individual patient including, among others, the size, weight, age and sex or disease condition of the patient, the nature and stage of the disease, and the route of administration”.

[0071] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the theragnostic agent of the present disclosure is administered in an amount from 40 to 200 mg Au@CeC>2 nanoparticles / kg body weight.

[0072] In an embodiment, optionally in combination with one or more features of the various embodiments described above, the theragnostic agent of the present disclosure is administered in an amount from 50 to 150 mg Au@CeC>2 nanoparticles / kg body weight.

[0073] As mentioned above, the theragnostic agent of the present disclosure can be prepared by a process comprising preparing a colloidal solution of the Au@CeC>2NPs and adding to the colloidal solution a pharmaceutically acceptable excipient for intravenous or intradermal administration.

[0074] In an embodiment, the process comprises: (a) preparing a initial colloidal solution of the Au@CeC>2NPs comprising an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core; (b) concentrating the initial colloidal solution obtained in step (a) until obtaining a concentrated colloidal solution comprising from 10 to 100 mg / mL, particularly from 15 to 50 mg / mL, more particularly from 20 to 40 mg / mL, Au@CeC>2NPs; and (c) adding to the concentrated colloidal solution the pharmaceutically acceptable excipients for intravenous or intradermal administration.

[0075] In an embodiment of the process for the preparation of the theragnostic agent, the CeC>2 shell has a thickness from 5 to 10 nm.

[0076] In an embodiment of the process for the preparation of the theragnostic agent, the CeC>2 shell has a thickness from 5 to 5.5 nm.

[0077] In an embodiment of the process for the preparation of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the Au core has an average diameter from 5 to 15 nm, particularly of 9-10 nm.

[0078] In an embodiment of the process for the preparation of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, wherein the CeC>2 shell comprises CeC>2 crystals of 2-3 nm in size. In an embodiment of the process for the preparation of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the nanoparticles have a size from 10 to 60 nm determined by TEM.

[0079] In an embodiment of the process for the preparation of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the nanoparticles have a size from 10 to 15 nm determined by TEM.

[0080] In an embodiment of the process for the preparation of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the concentrated colloidal solution comprises from 15 to 50 mg / mL Au@CeC>2NPs.

[0081] In an embodiment of the process for the preparation of the theragnostic agent, optionally in combination with one or more features of the various embodiments described above, the concentrated colloidal solution comprises from particularly from, more particularly from 20 to 40 mg / mL.

[0082] In a particular embodiment of the process above, the Au@CeC>2NPs can be synthesized by reacting in aqueous solution a cerium salt such as Ce(NOa)3 or CeCh, a gold salt such as HAuCL, and either citric acid or a citrate salt such as sodium citrate, and by adjusting the pH of the solution at a value from 7.5 to 8.5, particularly of 8. As an instance, they can be prepared as disclosed in the Example 1 herein below.

[0083] Hence, in an embodiment, optionally in combination with one or more features of the various embodiments described above, the initial colloidal solution is obtained in the presence of sodium citrate or citric acid, i.e., further comprises sodium citrate or citric acid.

[0084] In another embodiment, optionally in combination with one or more features of the various embodiments described above, the process further comprising adjusting the pH of the colloidal solution obtained in step (a) at a value from 7.5 to 8.5, particularly of 8.

[0085] In a particular embodiment, the process for the preparation of the theragnostic agent of the present disclosure comprises: (a) preparing a initial colloidal solution of the Au@CeC>2NPs by reacting in aqueous solution a cerium salt, a gold salt, and either citric acid or a citrate salt such as sodium citrate, and by adjusting the pH of the solution at a value from 7.5 to 8.5, particularly of 8; (b) concentrating the initial colloidal solution obtained in step (a) until obtaining a concentrated colloidal solution comprising from 15 to 50 mg / mL, more particularly from 20 to 40 mg / mL, Au@CeC>2NPs; and (c) adding to the concentrated colloidal solution the pharmaceutically acceptable excipients for intravenous or intradermal administration. In a particular embodiment, optionally in combination with one or more features of the various embodiments described above, the cerium salt is Ce(NOa)3 or CeCh, the gold salt is HAuCk In a more particular embodiment, optionally in combination with one or more features of the various embodiments described above, the reaction of step (a) is carried out in the presence of a citrate salt, particularly, of sodium citrate.

[0086] In another particular embodiment, the initial colloidal solution of the Au@CeC>2NPs can be prepared by a process comprising the following steps: a) providing an aqueous solution of a citrate salt at a temperature from . °C to

[0087] °C, such as 80 °C, and injecting a Ce(NOa)3 aqueous solution to obtain a first solution; b) injecting a HAuCU aqueous solution to the first solution at a temperature from 80 °C to lower than the boiling point of the solution; c) adjusting the pH of the solution at a pH from higher that 7 to lower than 9 and boiling the solution until completion of the reaction; d) cooling down the reaction media obtained in step c to room temperature in order to obtain a colloidal solution of Au@CeC>2NPs; c) optionally, isolate the Au@CeC>2NPs.

[0088] Concentration of the Au@CeC>2 NPs can be performed by centrifugation, removal of at least part of the supernatant, e.g. by decantation, and resuspension the remaining fraction in water. The NPs can be further concentrated by removing more solvent via controlled flow of N2 gas, particularly at room temperature.

[0089] Thus, in still another embodiment, optionally in combination with one or more features of the various embodiments described above, wherein the concentration step (b) is carried out by centrifugation, removal of at least part of the water, and further resuspension in water.

[0090] In another embodiment of the process of the present disclosure, the colloidal solution of step (a) has a initial volume, and step (b) is performed by centrifugation and subsequent reduction of the initial volume until a final volume from 10 to 15% of the initial volume.

[0091] In another embodiment, after centrifugation a supernatant is obtained, the supernatant is subsequently discarded to obtain a solid fraction, and the solid fraction is resuspended in water to obtain the concentrated colloidal solution.

[0092] In another embodiment, the process further comprises adjusting the final volume by evaporation of water at room temperature with the use of a continuous flow of a dry inert gas, particularly, dry N2.

[0093] It is also part of the present disclosure, a theragnostic agent obtainable by the process disclosed herein above.

[0094] The present disclosure also refers to the use of the Au@CeC>2 nanoparticles defined herein for the manufacture of an article of manufacture providing protection against ionizing radiation such as X-ray and ultraviolet, for instance, in radiation facilities in clinics in general, including dental clinics.

[0095] Also forms part of the invention an article of manufacture comprising the Au@CeC>2 nanoparticles disclosed herein. Particularly, the article of manufacture can be selected from the group consisting of coating materials, aprons, glasses, gloves, and collars.

[0096] In the present disclosure, it is noted that when discussing the process for the preparation of the Au@CeC>2 NPs defined above or the theragnostic agent of the present disclosure, each one of the embodiments or features defined for the Au@CeC>2 NPs or for the theragnostic agent can be considered applicable to the process of manufacture, when pertinent, whether or not they are explicitly discussed in the context of that other aspect.

[0097] 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”.

[0098] The following examples and drawings 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.

[0099] Examples

[0100] Materials

[0101] Cerium (III) nitrate hexahydrated (Ce(NC>3)3.6H2O), tetrachloroauric (III) acid HAuC SFW (99.9% purity), trisodium citrate dihydrate (>99%), tetramethylammonium hydroxide (TMAOH; 1M), sodium borohydride (NaBF ), sodium hydroxide (NaOH), 11- Mercaptoundecanoic acid (95%), hydrogen peroxide solution (30 % (w / w) in H2O) and Fetal Bovine Serum (FBS) (research grade, sterile filtered) were purchased from Sigma- Aldrich. Bovine Serum albumin (BSA) and Minimum Essential Medium (a-MEM) were purchased from ThermoFisher Scientific. PBS (Dulbecco's phosphate buffered saline 1x) was obtained from Biowest and Dulbecco’s Modified Eagle Medium, DMEM, was purchased from Capricorn Scientific. All reagents were used as received without further purification. Mili-Q water was used in the preparation of all solutions.

[0102] Characterization techniques

[0103] All NP suspensions were analyzed using a combination of techniques which are known to provide a reliable method to fully characterise the physicochemical properties of NPs.

[0104] UV-Visible Spectroscopy: UV-Vis spectra were recorded using a Cary 60 spectrophotometer from Agilent Technologies (USA). A 1 mL aliquot of the NP solution was carefully loaded into a cuvette, and spectral analysis was conducted across the 200- 800 nm wavelength range. Unless otherwise specified, H2O was employed as the baseline.

[0105] Dynamic Light Scattering (PLS) and -Potential Measurements: Measurements were conducted using a Malvern ZetaSizer Nano ZS instrument operating with a light source wavelength of 532 nm and a fixed scattering angle of 173° for DLS measurements. The software was configured with specific material parameters, including refractive index and, absorption coefficient. Solvent viscosity was set to that of H2O at 25 °C. Each value was the average of at least 3 independent measurements. DLS was used to determine the colloidal stability of the samples (given by a monomodal peak in the Intensity) and the hydrodynamic diameter (given by Number peak). The stability and surface charge of a colloidal solution of NPs was assessed by ^-potential, ^-potential values > 30 indicate colloidal stability by electrostatic repulsion forces. For ^-potential < 30, colloidal stability is predominantly through steric repulsion.

[0106] Transmission Electron Microscopy (TEM): TEM images were acquired with a JEOL 1010 transmission electron microscope (Tokyo, Japan) operated at low accelerating voltage (80 kV). For sample preparation, a 10 pl aliquot of the sample was carefully dropcast onto a carbon-coated copper grid and subsequently air-dried at room temperature. The analysis involved the examination of more than 500 individual particles, with size distribution determined using Imaged (National Institutes of Health, MD, USA).

[0107] ICP-OES: Elemental composition was determined by Inductively Coupled Plasma Optical Emission Spectroscopy (Perkin Elmer, Optima 4300DV). For sample preparation, 50 pL aliquots of the ultimately concentrated NP solution were carefully dissolved in 1 mL of aqua regia. Dissolution process was carried out using a microwave digestion oven (Milestone, Ultrawave).

[0108] Ce3+Concentration by Xylenol orange Test: The test performed is based on the protocol developed by Tonosaki et al. Xylenol orange, a metallochromic indicator, forms a 1 :1 complex with free Ce3+ions, quantifiable through UV-Vis spectrometry with an absorption peak at 575 nm. In brief, 10 pl of the sample is mixed with 700 pl of 1 mM Xylenol Orange, 3000 pl of acetate buffer (pH=6.0), and 1290 pl of MilliQ H2O in a vial. Injecting the sample induces a colour change in the solution that is more intense as the Ce3+concentration increases. Linear calibration curves were established for Ce3+concentrations ranging from 0 to 40 pg / ml. To determine Ce3+content in an unknown sample, the absorbance of the reagent blank is subtracted, and the calibration graph is utilized. The synthetic reaction yield of CeChNPs is assessed by analyzing the reaction's supernatant.

[0109] Nanoparticles synthesis and concentration processes

[0110] Clinical CT scans demand the administration of contrast agents at relatively high mass concentrations to achieve effective tissue contrast enhancement and obtain diagnostically valuable images. As a standard practice, contrast agents are evaluated at concentrations spanning from 200-400 mg / mL to ensure the attainment of clinically relevant attenuation values, generally falling between 0 and 1000 Hounsfield units (HU). These concentrations are chosen to achieve a high performance within biological tissues while avoiding adverse effects. NPs are typically synthesized in aqueous solvents at lower concentrations, ranging from 0.03 mg / mL (Au, ~10 nm, 3 x 1012NPs / mL) to 2.5 mg / mL (CeC>2, ~5 nm, 5 x 1015NPs / mL), corresponding to concentrations of 0.5 mM (Au) to 12 mM (Ce). Although the as-synthesized CeChNPs and Au@CeC>2NPs exhibit already considered high concentrations, they can be subsequently concentrated to achieve better attenuation values. Achieving the desired high NP concentration is a critical step since the colloidal stability of the sample may be compromised as NP concentration increases. This susceptibility to aggregation arises from the highly reactive nature of NP surfaces, making them prone to aggregation when exceeding typical working concentrations. Aggregation is a significant concern, as it can result in uncontrolled biodistribution within biological systems and trigger the sedimentation of NPs, potentially inducing undue oxidative stress.

[0111] To increase the NP concentration while preserving their colloidal stability, a two-step concentration process was implemented. In the first step, colloids were functionalized with biocompatible molecules, either post-synthesis (BSA for CeChNPs) or during synthesis (sodium citrate for Au@CeC>2NPs), to reduce high surface energy. The conjugation of as- synthesized CeChNPs with BSA molecules resulted in the formation of stable proteincoronas that confer long-term steric stability to the NPs, even at high concentrations. For Au@CeC>2NPs, the presence of sodium citrate imparts stability through electrostatic repulsion. Then, samples were concentrated by centrifugation to about 10-15% of their original volume. Final concentration adjustments were achieved in milder conditions since high NP concentrations are easy to destabilize and too much centrifugation makes the pellet non-reconstitutable. Thus, through room temperature evaporation using a continuous dry N2 flow, the excess solvent towards the final concentration was gently removed. Both colloidal solutions remained stable at high concentrations over an extended period of time (28 days) as indicated by UV-Vis spectra, DLS measurements, - potential, and TEM images. Gold nanoparticles (AuNPs), cerium oxide nanoparticles (CeChNPs) and cerium-gold hybrids nanoparticles (Au@CeC>2NPs) were synthesized and concentrated as disclosed herein below.

[0112] Synthesis of AuNPs (Comparative Example 1; control)

[0113] 200 mL of aqueous solution containing 0.25 mM HAuCL and 0.25 mM trisodium citrate were prepared. Then, 2.65 mL ice-cold freshly prepared NaBH4 (100 mM) was fastly added to the solution, while stirring at room temperature. The solution turned red immediately after the addition of NaBH4, indicating the formation of the nanoparticles. As the reaction took place at room temperature, citrate acted as stabilizer agent in this synthesis. The resultant particles (~4.5 nm, 7 1013NPs / mL) were negatively charged and well dispersed in double-distilled water.

[0114] After synthesis, AuNPs were at a concentration of 0.25 mM, what means that they have to be concentrated a ~103factor. Likewise CeChNPs, naked AuNPs have a very high surface energy and do not withstand being concentrated such a big factor without aggregation. Moreover, small AuNPs of 4.8 nm are hardly precipitated by centrifugation, and an alternative procedure should be applied. Accordingly with the characteristics of this system, we employed a conjugation-destabilization-resuspension method.

[0115] Concentration process of AuNPs (Comparative Example 2)

[0116] Firstly, AuNPs were conjugated to 11-mercaptoundecanoic acid (MUA). The amount of MUA needed to cover all the AuNPs was calculated from the footprint of MUA in Au, reported to be 0.21 nm. To ensure saturation coating, a 10-fold excess of the theoretical amount of MUA was added to the AuNPs. In detail, 120 ml of 10 mM MUA aqueous solution were added to 4 L of AuNPs solution and gently stirred for 24 h. Afterwards, MUA-coated AuNPs were precipitated by adding 200 ml of Glycine / HCI buffer (200 mM, 2.8 pH), which protonates MUA carboxylic acids. The resultant precipitate was centrifuged (7000 g, 15 min) to eliminate the acidic supernatant and resuspended in 10 ml of tricine buffer (50 mM, pH 8.0). AuNPs were further concentrated with a flow of N2 gas, as described above (0.5 L / min flow rate, -4 hours, mild stirring) until the desired final concentration was reached (-200 mM Au, ~1.6- 1016NPs / mL).

[0117] Successful adsorption of MUA molecules onto Au surface is inferred from the absorption spectra, as the initial SPR peak of naked NPs underwent a red-shift after MUA conjugation, from 505 nm to 518 nm. The formation of the MUA layer around the AuNPs is important as it provides electrostatic stability to the colloidal system (^-Potential: -61.1 ± 3.3 mV). Regarding DLS, it showed monomodal curves with an increased hydrodynamic diameter compared to the as synthesized NPs, indicative of MUA conjugation (Ad~2.3 nm). Subsequent precipitation with glycine acidic buffer and resuspension in tricine yielded well dispersed AuNPs: absorption spectra did not indicate aggregation, what was confirmed with DLS analysis, ^-potential, and TEM images. Final concentrated colloidal sample (193.9 ± 0.2 mM, by ICP-OES) was stored at 4 °C and remained stable over 48 hours. At longer times, physicochemical properties (UV-Vis, DLS, ^-Potential) did not show significant changes but some precipitation was observed. Thus, AuNPs were freshly concentrated just before the CT measurements.

[0118] Synthesis CeC NPs (Comparative Example 3)

[0119] Positively-charged CeChNPs of around 5 nm were synthesized by the chemical precipitation of cerium (III) nitrate hexahydrated (Ce(NC>3)3.6H2O) in a basic aqueous solution (37). 37.5 mM of cerium (III) nitrate hexahydrate was dissolved in 50 mL of Milli-Q water at room temperature. Then, 50 mL of tetramethylammonium hydroxide (TMAOH) solution 90 mM was added slowly at room temperature under vigorous stirring. Final stoichiometry Ce(NC>3)3:TMAOH was 1 :2.4. This stoichiometry ensured a high concentration of OH- ions facilitating the conversion of the cerium precursor while preventing its precipitation as solid Ce(OH)3. The mixture was left under soft stirring for 48 h until the reaction finished. NPs were purified by centrifugation (20000 g, 45 min) and the resultant pellet was resuspended in 80 mL aqueous solution of 1 mM TMAOH, which acts as a stabilizer. As determined by Xylenol Orange test, the reaction efficiency was 85.2% and the final cerium concentration 2.3 mg / mL (5.0- 1015NPs / mL; 18 mM Ce).

[0120] CeO2NPs exhibited a hydrodynamic diameter (DLS) of 13.6 ± 1.1 mV nm and a positive surface charge of + 57.7 mV at pH = 8, due to the TMA+ molecules adsorbed onto their surfaces (see Tables 1 and 2 below).

[0121] Concentration Process of CeChNPs (Comparative Example 4)

[0122] CeO2NPs were initially conjugated with Bovine Serum Albumin (BSA) to enhance colloidal stability. Specifically, 160 mg / ml of BSA was dissolved without stirring in 20 ml of 50 mM PB buffer and kept refrigerated. Subsequently, 80 ml of CeChNPs were gently added to the BSA solution with careful stirring. This controlled addition facilitated CeChNPs-BSA interaction, resulting in successful conjugation. The resulting solution, containing 100 ml of CeC>2:BSA, reached a concentration of 2.2:40 mg / ml, marking successful conjugation. To ensure thorough BSA adsorption onto CeChNPs' surface, the sample was refrigerated for 48 hours, allowing effective binding for enhanced colloidal stability. In the first concentration phase, the colloidal solution was concentrated via centrifugal filtration (4500 g for 15 minutes) using a 30 kDa molecular weight cutoff membrane (Millipore Amico Ultra filter), resulting in a concentration of -120 mM Ce (3.4 1016NPs / mL). The subsequent phase involved controlled solvent (H2O) evaporation using a continuous flow of N2 gas at 0.5 L / min over -3 hours. The sample was placed in close proximity to the N2 gas source and maintained under mild stirring conditions. The procedure allowed for precise adjustment of the final volume to attain a concentration of 140.9 mM Ce (4.2- 1016NPs / mL).

[0123] Negatively-charged Au@CeC>2NPs of ~20 nm at a concentration of 3 1013NPs / mL were produced following the following procedure:

[0124] 1000 mL of freshly prepared trisodium citrate (10 mM) was heated with a heating mantle in a 1 L three-necked round-bottom flask under vigorous stirring. Once the temperature reached 80 °C, 8.3 ml of Ce(N03)3 (100 mM) were injected (Cf =0.8 mM, 0.16 mg / mL). The solution turned yellowish after several seconds. Just before the solution started boiling, 8.3 ml of HAuCL aqueous solution 100 mM was quickly injected (Cf =0.8 mM, 0.11 mg / mL). The colour of the solution changed rapidly to black-grey and then it turned progressively to purple-red. pH of the solution was additionally adjusted with 20 mL of sodium hydroxide (NaOH, 100 mM) to ensure the solution remained at slightly basic pH (pH~8). The solution was kept boiling during 4 hours to ensure complete reaction of the precursors. The formation of NPs was carefully tracked using UV-Vis spectroscopy, revealing two key observations: a progressively intensifying and red-shifting of the surface plasmon resonance (SPR) peak over time, along with a gradual elevation of the cerium peak at 290 nm. After 4 hours there was no significant change in the spectra. Then the reaction was cooled down to room temperature. The resultant particles (0.27 mg / mL Au@CeC>2NPs) were colloidally stable and negatively charged.

[0125] Au@CeC>2NPs were composed of a central Au core of about 9 nm surrounded by a relatively uniform CeC>2 shell of about 5-5.5 nm composed of tiny CeC>2 crystals of 2-3 nm in size bound to the Au core. The heavy atom concentration in the NPs was 0.27 mg / mL. Au@CeC>2NPs exhibited a hydrodynamic diameter (DLS) of -37.8 ± 4.2 nm and a negative surface charge of -50.6 mV at pH = 8 due to the citrate molecules adsorbed onto their surfaces (Tables 1 and 2).

[0126] Concentration Process of AutcbCeC NPs (Example 2)

[0127] The use of sodium citrate during the synthesis conferred exceptional stability to Au@CeC>2 NPs, eliminating the necessity for additional surface modifications. For NP’s concentration, as-synthesized NPs were centrifuged at 16000 g during 30 minutes, the supernatant is discarded, and the remaining fraction (the pellet) is resuspended in H2O, resulting in a concentration of 20 mg / mL (-167 mM (Au+Ce), 2.7- 1015NPs / mL).

[0128] Tables 1 and 2 shows the characterization parameters of as-synthesized nanoparticles and after the concentration process. Table 1

[0129] Table 2

[0130] Cell Viability and in vivo biocompatibility

[0131] Cell Viability

[0132] Breast cancer cells (MCF-7) and HeLa cells were cultured in a-MEM supplemented with 10% FBS and 1% penicillin / streptomycin. Both cell lines were incubated in a humidified atmosphere containing 5% CO2 at a temperature of 37 °C. Cells were seeded onto 96- well plates at a density of 5000 cells / well and incubated for 24 hours to ensure proper cell attachment. All NPs were sterilized using a 0.2 pm syringe filter and were subsequently diluted to concentrations ranging from 1 to 100 pg / ml in the respective culture media. Following a 24-hour incubation with NPs, cell viability was assessed using the PrestoBlue® assay (Invitrogen, Life Technologies). Specifically, 10 pL of PrestoBlue® reagent was added to each well, and after a 2-hour incubation, fluorescence was measured (excitation: 530 nm, emission: 590 nm) using a BioTek Flx800 microplate reader. Cell cytotoxicity was quantified as a percentage relative to non-treated cells, with each exposure concentration tested in triplicate.

[0133] In vivo biocompatibility

[0134] In vivo applications were tested on healthy pregnant female Wistar rats provided by the Preclinical Validation group at VHIR when studying potential penetration of small nanoparticles into the placenta . CeChNPs were administered intravenously (200 microliters of a 17 mg / ml Au@CeO2 solution). Images were obtained at 70 kVp, 160 pA, 60 x 60 cm FOV, and 4.5 minutes per scan (data not shown). Accumulation of cerium at the end (30 days) of the experiment was quantified by mass spectroscopy and showed in Fig 1. No weight loss or altered behavior or other sign of toxicity was observed in the treated animals.

[0135] Amplex Red Hydrogen Peroxide Scavenging Assay

[0136] NPs ability to scavenge H2O2 was evaluated using the Amplex® Red Assay (Invitrogen, cat n° A22188). Both CeChNPs and AuNPs were tested under uniform conditions, with NPs buffer as a control. The Amplex Red reagent reacts with H2O2 in a 1 :1 ratio, yielding the red-fluorescent product resorufin. H2O2 samples were prepared in phosphate buffer (PB) with concentrations ranging from 0 to 6 pM. Next, 10 pl of NPs solution (0.5 mg / ml) was added, and the mixture incubated for 10 minutes. Subsequently, 20 pl of Amplex Red reagent (250 pM) and 10 pl of horseradish peroxidase (HRP) enzyme (1 ll / rnl) were introduced, bringing the final reaction volume to 100 pl per well. Following a 30-minute dark incubation, fluorescence emission was measured using a Multi-Detection Microplate Reader (FL800, BioTek® Instruments, Inc). Excitation occurred at 530 ± 20 nm, with fluorescence detection at 590 ± 20 nm. Background fluorescence was subtracted using a H2O2 -free control. A calibration curve was employed to quantify H2O2 concentrations in each sample. This procedure was conducted in triplicate, with average values provided.

[0137] CT Contrast Experiments

[0138] Quantitative CT measurements were conducted using a micro-CT scanner (Quantum FX, Perkin Elmer). Various tube energies (50, 70, and 90 kVp) were tested, each with an X- ray tube current of 160 pA. The field of view was set at 60 x 60 cm, and 256 projections were acquired during a 4.5-minute scan, achieving a maximum resolution of 1024 x 1024 pixels The field of view was set at 60 x 60 cm, and 256 projections were acquired during a 4.5-minute scan, achieving a maximum resolution of 1024 x 1024 pixels. Image analysis was performed using the free software Amide. CT attenuation values were provided in Hounsfield units (HU) after calibration with water (0 HU) using the formula: HU = 100. (p - pH2O) / pH2O, where p and PH2O represent the linear X-ray attenuation coefficients of the material and H2O, respectively. These experiments were conducted in triplicate.

[0139] In vivo applications were tested on male athymic nude mice with sarcoma tumors (10-day- old), provided by the Preclinical Validation group at VHIR. CeO2NPs were administered intratumorally (10 mg Ce / ml Ce; 75 pl), and images were obtained at 70 kVp, 160 pA, 60 x 60 cm FOV, and 4.5 minutes per scan.

[0140] Results

[0141] The CT contrast properties of CeO2NPs and Au@CeO2NPs were investigated using a clinical CT scanner. Quantitative CT measurements were conducted using a micro-CT scanner Quantum FX. Various tube energies (50, 70, and 90 kVp) were tested, each with an X-ray tube current of 160 pA. CT attenuation values were provided in Hounsfield units (HU) after calibration with water (0 HU). In vitro, CT contrast efficiency was assessed by comparing the X-ray absorption of the different NPs to that produced by the commercial iodinated product lohexol (Omnipaque 300 mg lodine / mL). lohexol, an EMA and FDA- approved CT contrast agent, together with AuNPs, served as controls, all evaluated at equimolar concentrations of heavy atoms. The solutions of the different materials were prepared in concentrations ranging from 0 to 200 mM and placed in a 96-well microplate (200 pl / well). Control ionic substances (Ce(NO3)3, HAuCU) and lohexol were diluted in MilliQ H2O. CeO2NPs were diluted in PB buffer (10 mM, pH 7.6), and Au@CeO2NPs in tricine buffer (50 mM, pH 8) to maintain colloidal stability during dilution. These experiments were conducted in triplicate.

[0142] The CT attenuation exhibited a linear correlation with the mass concentration of the NPs (Ce or Au) at each tube voltage employed (Fig. 1A-C). The extracted CT attenuation rates of the various agents (CeO2NPs, Au@CeO2NPs, AuNPs, and lohexol) at 50,70 and 90 kVp incident tube potential are depicted in Table 3.

[0143] Table 3

[0144] Our findings illustrate that both Ce02 and Au@CeO2NPs deliver substantial contrast enhancement across all three tested tube potentials: 50 kVp, 70 kVp, and 90 kVp (Fig. 1 D-F). Significantly, at 50 kVp, the X-ray attenuation of Ce approaches that of lohexol, falling well within the measurable range of the CT equipment. At 70 kVp and 90 kVp tube potentials, CeO2NPs exhibited a substantial enhancement in contrast, effectively competing with the attenuation capabilities of the iodinated agent. This effect arises because iodine possesses a slightly lower K-edge value (33.2 keV) than cerium. Notably, the incorporation of an Au core within the NPs (Au@CeC>2NPs) further increases CT contrast at all energy levels, with the most significant enhancement observed at 50 kVp compared to the iodinate agent. In the case of AuNPs, the CT attenuation rate increases with higher tube voltage. Indeed, each type of NP optimizes image contrast at distinct incident tube potentials.

[0145] CeChNPs effectively enhance X-ray contrast when the maximum peak of the incident X- ray spectrum falls within the energy range of 40.4 keV to 80.7 keV. This energy range typically corresponds to intermediate tube potentials, usually within the range of 60-100 kVp, commonly used in clinical radiography and pediatric CT scans. Surprisingly, in cases where the spectrum peak is at lower energies than 40 keV (around 30-60 kVp, typical for mammography) or higher energies than 81 keV (approximately 110-140 kVp, typical for clinical CT), Au@CeC>2NPs excel in enhancing X-ray contrast.

[0146] For the sake of comparison, phantoms were prepared for imaging of the NPs at the three different tube voltages (Fig. 2H-J). Phantoms obtained with the corresponding ionic compounds at the same concentrations are shown in Figure 3.

[0147] The impact of aggregation by conducting contrast enhancement tests on a mixture of poorly dispersible (aggregated) CeChNPs and AuNPs was also evaluated. This was then compared to equimolar concentrations of Ce(NC>3)3 and HAuCU salts, which disperse uniformly. It could be seen that while the contrast intensity per total area is similar, the distribution of contrast exhibited significant variations, resulting in the generation of artifacts in the CT image. Consequently, significant discrepancies were observed between the triplicate experiments, leading to large error bars. In contrast, Au@CeC>2NPs hybrids exhibited superior colloidal stability, enabling easier manipulation and yielding accurate CT attenuation values. In a visual demonstration, this effective contrast enhancement is illustrated through a phantom sheet created using a 3D printer, engraved with chemical element symbols, and filled with solutions containing the respective CeChNPs and Au@CeC>2NPs solutions. The resulting X-ray image obtained using p-CT (70 kVp, 200 pA) is presented in Figure 2K.

[0148] Colloidal stability

[0149] Colloidal stability in physiological media of NPs was tested through incubation at 37 °C in complete cell culture medium (cCCM), consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). The NPs were mixed with cCCM at a concentration ratio of 1 :10 by volume under sterile conditions and placed in an incubator for different incubation times. This specific NPs:cCCM ratio was chosen to optimize cell viability while avoiding excessive dilution of the cCCM, which could compromise cell survival. Sample stability was monitored over time by UV-Vis spectroscopy, DLS, and potential measurements, before and after purification (centrifugation at 15,000 g for 20 minutes, followed by resuspension in 1 mL of H2O). The results showed the sustained stability of both CeChNPs and Au@CeC>2NPs over an extended duration, with observations extending up to 14 days.

[0150] ROS scavenger capacity

[0151] H2O2 was used as a ROS precursor to evaluate the antioxidant capacity of CeO2NPs and Au@CeO2NPs using the Amplex Red assay (Fig. 4A-B). As shown, at equal mass concentrations, Ce-based NPs induce a pronounced scavenging of H2O2. In contrast, AuNPs are not able to reduce H2O2 control levels, as expected. Surprisingly, the activity of the Au@CeO2NPs was found to be higher than that of pure CeO2NPs. These results suggest a synergetic effect on the antioxidant activity produced probably by the Au-CeO2 interfaces. Amplex Red tests were performed on both as-synthesized and functionalized NPs to assess the impact of surface functionalization. Figure 4C shows how surface molecules have minimal influence on the results, as similar values are obtained in comparison. For the sake of reproducibility, two sets of Au@CeC>2NPs were synthesized and analyzed with Amplex Red under identical conditions, showing no significant difference indicating the robustness of the synthesis and concentration processes (Fig. 4D). We also conducted comparative dose-response cytotoxicity studies using CeChNPs and Au@CeC>2NPs. Cell viability was tested on MCF-7 and HeLa cells (Fig. 4E-F). Breast cancer cells (MCF-7) and HeLa cells were cultured in DMEM with 10% FBS and 1% penicillin / streptomycin in a 37°C, 5% CO2 environment. After 24 hours of incubation to ensure cell attachment, they were seeded into 96-well plates at 5000 cells / well. Nanoparticles were sterilized with a 0.2 pm syringe filter, and then diluted to concentrations of 1 to 100 pg / mL in the respective culture media. After another 24-hour incubation, cell viability was assessed with the PrestoBlue® assay. This involved adding 10 pL of PrestoBlue® reagent to each well, followed by fluorescence measurement (excitation: 530 nm, emission: 590 nm) using a microplate reader. Cytotoxicity was expressed as a percentage relative to non-treated cells, with each concentration tested in triplicate. Encouragingly, none of the NPs exhibited significant toxicity at the employed concentrations. These findings were corroborated by fluorescence and optical microscope images, which demonstrated the absence of substantial cell detachment.

[0152] Biodistribution assay

[0153] A Biodistribution assay was carried out for tracking where compounds of interest travel in an experimental animal. Thus, in vivo applications were tested on healthy female Wistar rats provided by the Preclinical Validation group at the Fundacio Hospital Universitari Vail d’Hebron - Institut de Recerca (VHIR). AuCeChNPs were administered intravenously (200 microliters of a 17 mg / ml Au@CeO2 solution). Images were obtained at 70 kVp, 160 pA, 60 x 60 cm FOV, and 4.5 minutes per scan (data not shown). Accumulation of Au and Ce at the end of the experiment was quantified by mass spectroscopy. The results confirmed that the contrast enhancement was finely correlated with elemental analysis using calibration data (HU vs concentration). As expected, there was a good correlation between the in vivo intensity contrast and elemental concentration.

[0154] Comparative Examples 5 and 6

[0155] Comparative Example 5 - Synthesis of Au-core CeO2-shell Nanoparticles disclosed in Jain V, et al.

[0156] Synthesis of Au Seed Nanoparticles. Briefly, 5 mL of HAuCI43H2O (10 mM) was mixed with 0.5 mL of CTAB (0.2 mM) under stirring for 10 minutes. Then, 0.6 mL of freshly prepared NaBH4(0.1 M) was added as the reducing agent. Upon addition, the solution turned brown, indicating the formation of gold nanoparticles (AuNPs). After overnight incubation at room temperature, the solution developed a red color, confirming seed formation. The Au seed solution was aged for 96 hours before use in the synthesis of Au- core CeO2-shell nanoparticles.

[0157] Synthesis of Au-core CeO2-shell Nanoparticles. To prepare the Au-core CeO2-shell nanoparticles, 0.8 mL of the aged CTAB-coated Au seed solution was mixed with 7.2 mL of CTAB (0.025 M) and 0.8 mL of EDTA NH3solution. Then, a Ce(NO3)36H2O solution (final concentration: 0.6 mM) was added to reach a final volume of approximately 10 mL. The resulting suspension was mixed gently for 1 minute and then placed in a hot air oven at 90 °C for 5 hours. The nanoparticles were collected by centrifugation at 10,000 rpm for 5 minutes, and the pellet was redispersed in 100 mL of Milli-Q water for further use.

[0158] Comparative Example 6 - Synthesis of AuNR@CeO2Core-Shell Nanoparticles disclosed in Zhang W et al.

[0159] Synthesis of Au Seed Nanoparticles. Gold (Au) seed nanoparticles were synthesized by mixing 5 mL of HAuCI4(0.5 mM) with 0.5 mL of CTAB (0.2 mM) under vigorous stirring. To this solution, 0.6 mL of freshly prepared NaBH4(0.01 M, ice-cold) was rapidly added as the reducing agent. The solution immediately turned brown, indicating seed formation. The resulting dispersion was kept at room temperature for at least 2 hours before use in the growth of gold nanorods.

[0160] Synthesis of Gold Nanorods (AuNRs). To synthesize gold nanorods, 5 mL of CTAB (0.2 M) was mixed with 0.05 mL of AgNO3(4 mM), followed by the addition of 5 mL of HAuCI4(1 mM). After gentle mixing, 70 pL of freshly prepared ascorbic acid (78.8 mM) was added, resulting in a colorless solution. Then, 12 pL of the previously prepared Au seed solution was introduced to initiate nanorod growth. The reaction mixture was left undisturbed at RT for at least 12 hours. After growth, 10 mL of the AuNRs suspension was centrifuged at 11 ,000 rpm for 30 minutes. The pellet was collected and redispersed in Milli-Q water for subsequent use.

[0161] Synthesis of AuNR@CeO2Core-Shell Nanoparticles. For the growth of the CeO2shell, 8 mL of CTAB (0.025 M) was mixed with 0.8 mL of EDTA-NH3buffer solution (prepared by combining 20 mL of EDTA 0.025 M with 0.38 mL of 28% NH3). Then, 80 pL of Ce(NO3)3(0.1 M) was added under gentle stirring, followed by the AuNR dispersion. Immediately afterward, 1 mL of freshly prepared NaBH4(0.01 M) was added as the reducing agent, replacing hydrazine due to its limited commercial availability and significant handling restrictions associated with high toxicity and regulatory constraints. Sodium borohydride provides comparable reducing power under alkaline conditions and is widely adopted as a safer, more accessible alternative, offering lower toxicity, broader availability, and easier handling. Although hydrazine is a strong reducing agent, its volatility and hazardous nature limit its practical use in routine laboratory-scale syntheses. The mixture was stirred briefly heated at 90 °C for 5 hours. The resulting AuNR@CeO2nanoparticles were collected by centrifugation at 5,500 rpm for 30 minutes, washed, and redispersed in 10 mL of Milli-Q water for further use.

[0162] Experimental protocol followed to evaluate ROS scavenging activity Hydrogen peroxide (H2O2) concentrations were quantified using the Amplex® Red Hydrogen Peroxide / Peroxidase Assay Kit (Catalog No. A22188, Invitrogen, Thermo Fisher Scientific), which provides all necessary reagents: Amplex Red reagent, dimethyl sulfoxide (DMSO), 1X reaction buffer, horseradish peroxidase (HRP), and H2O2standards.

[0163] To prepare the working solution, 50 pL of 10 mM Amplex Red reagent was mixed with 100 pL of HRP (10 U / rnL) and 4.85 mL of 1X reaction buffer, yielding a total volume of 5 mL. The solution was freshly prepared and protected from light prior to use.

[0164] To assess the catalytic activity of the nanoparticles, 15 pM of H2O2was mixed with nanoparticle suspensions at 75 pM of elemental cerium in all cases in 1X reaction buffer. At selected timepoints, the residual H2O2concentration was determined. The mixtures were incubated in the dark at room temperature for 30 minutes to allow enzymatic conversion of Amplex Red into resorufin.

[0165] The absorbance of resorufin — directly proportional to the remaining H2O2concentration — was measured using a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, USA). Quantification was performed by interpolation from a calibration curve generated using H2O2standards ranging from 0 to 25 pM, ensuring measurements remained within the assay's linear and reliable detection range to avoid over-oxidation of resorufin to non- fluorescent products.

[0166] Results

[0167] H2O2 scavenging capacity of the Au@CeO2NPs of Example 1 was compared with the catalytic activity of NPs of Comparative Example 5 and NPs of Comparative Example 6. The results are shown in Fig. 5. All nanoparticle suspensions were tested at a concentration of 75 pM of elemental cerium to ensure comparability of catalytic activity.

[0168] It can be seen that Au@CeO2NPs of Example 1 show a significantly higher H2O2scavenging capacity than the one of Comparative Example 5 and the ones of Comparative Example 6. Therefore, Au@CeO2NPs of Example 1 are particularly effective when used in simultaneous medical diagnosis and radio-protection.

[0169] Citation List

[0170] 1. Garcia A, et al. (2023) Nanoceria as Safe Contrast Agents for X-ray CT Imaging". Nanomaterials (Basel). Vol. 13(15): 2208.

[0171] 2. Jain V, et al. (2019). Unveiling the effect of 11-MUA coating on biocompatibility and catalytic activity of a gold-core cerium oxide-shell-based nanozyme. RSC Advances. Vol. 9: 33195-33206. 3. Zhang et al. (2022). Emerging nanotherapeutics alleviating rheumatoid arthritis by readjusting the seeds and soils. Journal of Controlled Release. Vol. 345: 851-879.

[0172] 4. CN118001394.

[0173] 5. Tonosaki, K. et al. (1962). Spectrophotometric Determination of Cerium(lll) and Some Rare Earths with Xylenol Orange. Bull. Chem. Soc. Jpn. Vol 35 (10): 1683-1686

[0174] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses:

[0175] Clause 1. Au@CeO2 nanoparticles comprising an Au core having an average diameter from 5 to 100 nm and a CeO2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeO2 shell completely covers the surface of the Au core, for use in simultaneous diagnosis and radio-protection.

[0176] Clause 2. The Au@CeC>2 nanoparticles for use of clause 1 , wherein the nanoparticles have a size from 10 to 60 nm, determined by TEM.

[0177] Clause 3. A theragnostic agent comprising the Au@CeC>2 nanoparticles as defined in clauses 1 or 2 and pharmaceutically acceptable excipients.

[0178] Clause 4. The theragnostic agent of clause 3, wherein the pharmaceutically acceptable excipients comprise water for injection and sodium citrate or citric acid, and the theragnostic agent is in the form of a colloidal solution.

[0179] Clause 5. The theragnostic agent of clauses 4, wherein the Au@CeC>2 nanoparticles are at a concentration from 10 to 100 mg / mL.

[0180] Clause 6. The theragnostic agent of any one of clauses 3 to 5, for use in radio-guided microsurgery applications.

[0181] Clause 7. The theragnostic agent of any one of clauses 3 to 5, for use in simultaneous medical diagnosis and radio-protection, for instance, in dentistry, wherein medical diagnosis is performed by X-ray imaging.

[0182] Clause 8. The theragnostic agent for use of clause 7, wherein X-ray imaging is selected from the group consisting of contrast-enhanced mammography, urography, arteriography, phlebography, cardio angiography, cervical myelography, arthrography, hysterosalpingography, sialography, fluoroscopy, chest imaging, abdominal imaging, and paediatric imaging.

[0183] Clause 9. The theragnostic agent of clauses 7 or 8, wherein X-ray imaging is performed by X-ray computed tomography. Clause 10. The theragnostic agent of any one of clauses 7 to 9, wherein medical diagnosis comprises visualization of abnormal structures or lesions and differentiation between healthy and pathological tissue in a mammal, including a human.

[0184] Clause 11. The theragnostic agent of any one of clauses 3 to 10, wherein the theragnostic agent is either for intravenous or for intradermal administration.

[0185] Clause 12. The theragnostic agent of any one of clauses 3 to 11, wherein the theragnostic agent is administered in an amount from 40 to 200 mg / kg body weight.

[0186] Clause 13. A process for the preparation of a theragnostic agent comprising the Au@CeC>2 nanoparticles as defined in clauses 1 or 2, and pharmaceutically acceptable excipients, the process comprising:

[0187] (a) preparing a colloidal solution of the Au@CeC>2 nanoparticles;

[0188] (b) concentrating the colloidal solution obtained in step (b) until obtaining a concentrated colloidal solution comprising from 10 to 100 mg / mL Au@CeC>2 nanoparticles; and

[0189] (c) adding to the concentrated colloidal solution the pharmaceutically acceptable excipients for intravenous or intradermal administration.

[0190] Clause 14. Use of the Au@CeC>2 nanoparticles as defined in clauses 1 or 2 for the manufacture of an article of manufacture providing protection against ionizing radiation.

[0191] Clause 15. An article of manufacture comprising the Au@CeC>2 nanoparticles as defined in clauses 1 or 2.

Claims

Claims1 . Au@CeC>2 nanoparticles consisting of an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core, for use in simultaneous imaging diagnosis and radio-protection.

2. The Au@CeC>2 nanoparticles for use of claim 1 , wherein the CeC>2 shell has a thickness from 5 to 10 nm.

3. The Au@CeC>2 nanoparticles for use of claim 1 , wherein the CeC>2 shell has a thickness from 5 to 5.5 nm.

4. The Au@CeC>2 nanoparticles for use of any one of claims 1 to 3, wherein the Au core has an average diameter from 5 to 15 nm, particularly of 9-10 nm.

5. The Au@CeC>2 nanoparticles for use of any one of claims 1 to 3, wherein the CeC>2 shell comprises CeC>2 crystals of 2-3 nm in size.

6. The Au@CeC>2 nanoparticles for use of any one of claims 1 to 5, wherein the nanoparticles have a size from 10 to 60 nm, determined by TEM.

7. A theragnostic agent comprising i) Au@CeC>2 nanoparticles comprising an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core; and ii) pharmaceutically acceptable excipients; wherein the theragnostic agent is in the form of an colloidal solution, and wherein the Au@CeC>2 nanoparticles are at a concentration from 10 to 100 mg / mL; wherein the theragnostic agent is a dual-purpose radio-protecting and contrast agent.

8. The theragnostic agent of claim 7, wherein the CeC>2 shell has a thickness from 5 to 10 nm.

9. The theragnostic agent of claims 7 o 8, wherein the CeC>2 shell has a thickness from 5 to 5.5 nm.

10. The theragnostic agent of any one of claims 7 to 9, wherein the Au core has an average diameter from 5 to 15 nm, particularly of 9-10 nm.11 . The theragnostic agent of any one of claims 7 to 10, wherein the CeC>2 shell comprises CeC>2 crystals of 2-3 nm in size.

12. The theragnostic agent of any one of claims 7 to 11 , wherein the nanoparticles have a size from 10 to 60 nm determined by TEM.

13. The theragnostic agent of any one of claims 7 to 11 , wherein the nanoparticles have a size from 10 to 15 nm determined by TEM.

14. The theragnostic agent of of any one of claims 7 to 13, wherein the pharmaceutically acceptable excipients comprise water for injection and sodium citrate or citric acid.

15. A theragnostic agent wherein the theragnostic agent is for use in simultaneous imaging diagnosis and radio-protection, the theragnostic agent comprising i) Au@CeC>2 nanoparticles comprising an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core; ii) and pharmaceutically acceptable excipients; wherein the theragnostic agent is in the form of an colloidal solution; and wherein the Au@CeC>2 nanoparticles are at a concentration from 10 to 100 mg / mL; wherein the theragnostic agent is a dual-purpose radio-protecting and contrast agent.

16. The theragnostic agent for use of claim 15, wherein the pharmaceutically acceptable excipients comprise water for injection and sodium citrate or citric acid.

17. The theragnostic agent for use of claims 15 or 16, wherein the simultaneous imaging diagnosis and radio-protection is used in radio-guided microsurgery applications.

18. The theragnostic agent for use of claim 15 or 16, wherein medical diagnosis is performed by X-ray imaging.

19. The theragnostic agent for use of claim 18, wherein the X-ray imaging is used in dentistry.

20. The theragnostic agent for use of claims 18 or 19, wherein X-ray imaging is selected from the group consisting of contrast-enhanced mammography, urography, arteriography, phlebography, cardio angiography, cervical myelography, arthrography, hysterosalpingography, sialography, fluoroscopy, chest imaging, abdominal imaging, and paediatric imaging.21 . The theragnostic agent for use of any one of claims 18 to 20, wherein X-ray imaging is performed by X-ray computed tomography.

22. The theragnostic agent for use of any one of claims 18 to 21 , wherein medical diagnosis comprises visualization of abnormal structures or lesions and differentiation between healthy and pathological tissue in a mammal, including a human.

23. The theragnostic agent of any one of claims 7 to 14 or the theragnostic agent for use of any one of claims 15 to 22, wherein the theragnostic agent is either for intravenous or for intradermal administration.

24. The theragnostic agent of any one of claims 7 to 14 or the theragnostic agent for use of any one of claims 15 to 23, wherein the theragnostic agent is administered in an amount from 40 to 200 mg Au@CeC>2 nanoparticles / kg body weight.

25. A process for the preparation of a theragnostic agent as defined in claim 7, the process comprising:(a) preparing an initial colloidal solution of Au@CeC>2 nanoparticles comprising an Au core having an average diameter from 5 to 100 nm and a CeC>2 shell having a thickness from 5 to 20 nm, wherein the Au core have a surface, and the CeC>2 shell completely covers the surface of the Au core;(b) concentrating the initial colloidal solution obtained in step (a) until obtaining a concentrated colloidal solution comprising from 10 to 100 mg / mL Au@CeC>2 nanoparticles; and(c) adding to the concentrated colloidal solution the pharmaceutically acceptable excipients for intravenous or intradermal administration.

26. The process of claim 25, wherein the Au@CeC>2 nanoparticles are synthesized by reacting in aqueous solution a cerium salt, a gold salt, and either citric acid or a citrate salt, and by adjusting the pH of the solution at a value from 7.5 to 8.5.

27. The process of claims 25 or 26, wherein the colloidal solution of step (a) has a initial volume, and step (b) is performed by centrifugation and subsequent reduction of the initial volume until a final volume from 10 to 15% of the initial volume.

28. The process of claim 27, wherein after centrifugation a supernatant is obtained, the supernatant is subsequently discarded to obtain a solid fraction, and the solid fraction is resuspended in water to obtain the concentrated colloidal solution.

29. The process of claims 27 or 28, wherein the process further comprises adjusting the final volume by evaporation of water at room temperature with the use of a continuous flow of a dry inert gas, particularly, dry N2.

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