WO2021148973A1

WO2021148973A1 - Multifunctional nanoparticles based on metallic nanoalloys for diagnostic and therapeutic use

Info

Publication number: WO2021148973A1
Application number: PCT/IB2021/050449
Authority: WO
Inventors: Vincenzo AMENDOLA; Veronica TORRESAN; Andrea Guadagnini; Pasquina Marzola; Antonello SPINELLI
Original assignee: Universita' Degli Studi Di Padova; Università Degli Studi Di Verona; Ospedale San Raffaele S.R.L.
Priority date: 2020-01-21
Filing date: 2021-01-21
Publication date: 2021-07-29
Also published as: IT202000001048A1

Abstract

The present invention relates to nanoparticles comprising a metal nanoalloy and a biocompatible organic stabilizer, useful in the diagnosis and therapy of various pathological conditions, including cancer. Indeed, the proposed alloy nanoparticles possess the chemical and physical characteristics necessary to act as contrast agents for some diagnostic techniques based on the acquisition of three-dimensional images of the human body, such as nuclear magnetic resonance imaging (MRI) and X-ray computed axial tomography (CT), and to act as sensitizers for some radiotherapy techniques such as radiotherapy using X-rays (XRT) or neutron capture (NCT). In some of the embodiments of the invention, these characteristics can coexist in the same metal nanoalloy. The same nanoalloys spontaneously reduce their size over time to facilitate clearance.

Description

TITLE

Multifunctional nanoparticles based on metallic nanoalloys for diagnostic and therapeutic use.

DESCRIPTION

FIELD OF THE INVENTION

The present invention relates to nanoparticles comprising a metal nanoalloy and a biocompatible organic stabilizer, useful in the diagnosis and therapy of various pathological conditions, including cancer. This is due to the fact that the proposed alloy nanoparticles possess the chemical and physical characteristics necessary to act as contrast agents for some diagnostic techniques based on the acquisition of three- dimensional images of the human body, such as nuclear magnetic resonance imaging (MRI) and X-ray computed axial tomography (CT), and to act as sensitizers for some radiotherapy techniques such as X-ray radiotherapy (XRT) or neutron capture therapy (NCT). In some of the embodiments of the invention, such characteristics can coexist in the same metal nanoparticle. The nanoalloys of the invention are characterized by a dimension compatible with the optimal size threshold for the removal of foreign bodies from the organism, or by the ability to spontaneously degrade over time, reducing their size down to this size threshold.

In particular, the invention relates to nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprising a metal nanoalloy and a biocompatible organic stabilizer in which a) the nanoalloy essentially consists of X-Y-Z, in where X, Y and Z are different from each other and where X can be gold and / or silver or iron and / or manganese, Y can be iron, manganese or boron and Z can be silver, manganese, boron or it can be absent; and where b) when Z is present, X varies in an atomic percentage range between 5% and 95%, Y varies in an atomic percentage range between 2.5% and 85% and Z varies in an atomic percentage range between 2.5% and 50%; when Z is absent, X and Y vary in an atomic percentage range between 5% and 95% with the condition that when X is gold and Y is iron, X is present in atomic percentage between 15% and 80% and Y is present in atomic percentage between 20% and 85%.

The invention also refers to the combined, simultaneous and / or spatially coincident use of the nanoparticles of the invention as contrast agents in diagnostic techniques based on the acquisition of images of the human and / or animal body, such as for example for MRI and / or CT, and as sensitizing agents in radiotherapy techniques such as XRT and / or NCT.

BACKGROUND OF THE INVENTION

Nanomaterials (NMs) promise to revolutionize current diagnostic and therapeutic procedures, with a special focus on cancer treatment. In fact, while NMs are the object of intense research aimed at the creation and testing of new "nanomedicines", only a small part of the thousands of nanomedicines proposed to date are progressing towards clinical applications.

One of the limiting factors is the "nanomedicine dilemma": only nanoparticles with a size of tens of nanometers accumulate efficiently at target sites without the need to administer a large excess of the drug, while only very small NMs are rapidly eliminated from the body in a reasonably short time to minimize the interaction with the tissues and the immune system. Ideally, nanomedicines should behave like a 4-dimensional (4D) material, capable of reducing the size only after use, when it should be removed from the body.

Another limiting factor is the need to have nanomedicines capable of performing multiple functions. In particular, the availability of a single substance capable of carrying out a combined action of diagnosis and therapy avoid the repeated administration of distinct substances that can assist in, respectively, diagnosis and therapy, thus avoiding to duplicate the problems connected to duration, timing and method of administration, the tolerability of the administered compound and the absence of adverse reactions.

Furthermore, the awareness-raising action for the therapeutic treatment occurs in the same place where the contrast increase by diagnostic technique has been measured, providing also a quantitative indication of the sensitizer accumulation in the areas to treat.

This immediately results in calibration of the radiation dose to be used for the therapy, point by point in the area to be treated. Where such multifunctionality also includes the possibility of increasing the contrast for two different visualization techniques (MRI and CT), the additional advantage is the combination of an imaging technique selective for low-density tissues (MRI) with another selective for high density tissues (CT), in exactly the same area, and without duplicating the administration of contrast agents for the two different techniques.

Where such multifunctionality includes, in addition to the contrast for MRI and / or CT, also the sensitization for a radiotherapy technique (for example XRT or NCT), the additional advantage is that of locating the sensitizer precisely within the human body, quantifying its presence in a more accurate way than what is possible with molecular sensitizers and avoiding to give further high doses of drug, having all the functions already coexisting in the same compound.

Where this multifunctionality also extends to sensitization for two different radiotherapy techniques (XRT and NCT), the additional advantage is to perform combined treatments to maximize efficacy, reducing the overall dose of ionizing radiation compared to single treatments, while obtaining the same efficacy with lower side effects.

The use of nanomedicines, or compounds with dimensions between hundreds and a few nanometers, is a strategy frequently used to obtain multiple diagnostic and therapeutic functions in the same compound (D. Kim et al, Recent Development of Inorganic Nanoparticles for Biomedical Imaging, ACS Cent. Sci. (2018), vol. 4, pag. 324-336; X. Han et al, Applications of nanoparticles in biomedical imaging, Nanoscale (2019), vol. 11, p. 799; G. Yang et al, Degradability and Clearance of Inorganic Nanoparticles for Biomedical Applications, Adv. Mater. (2019), vol. 31, pag. 1805730; e Q. Chen et al., Nanoparticle-Enhanced Radiotherapy to Trigger Robust Cancer Immunotherapy, Adv. Mater. (2019), vol. 31, pag. 1802228).

Regarding bio-imaging, i.e., the visualization of biological structures and processes, a variety of techniques with specific advantages and limitations have been developed to meet the needs of various clinical and laboratory contexts. Bio-imaging techniques can non-invasively measure biological functions, evaluate cellular and molecular events, and reveal the internal functioning of a body. Examples of biological imaging techniques include MRI, positron emission tomography (PET), CT, luminescence (optical imaging), and ultrasound (US) (D. Kim et al. And X. Han et al., above mentioned).

MRI is a very powerful and widely used imaging modality. It is based on the properties of the spin of the proton when it is excited with a radiofrequency pulse in the presence of an external magnetic field. Magnetic resonance imaging provides high spatial and temporal resolution, and excellent intrinsic contrast of so-called "soft", i.e., low density, tissues. It also can show anatomical tomographic information. Furthermore, MRI does not use ionizing or radiotracer radiation. On the other hand, costs, longer imaging times, artefacts due to patient movement and potential artefacts due to prostheses and implants must be considered among the limits of magnetic resonance.

To solve some of these problems, contrast agents are used which significantly aid in the detection and differentiation of lesions from healthy tissues (X. Han et al., cited above). Clinical MRI contrast agents are based on gadolinium complexes, i.e. Gd(III) complexes, but nanostructured iron oxides have also been used. A serious side effect of Gd(III) complexes is the nephrogenic systemic fibrosis, while iron oxides are generally considered benign and biologically tolerable (D. Kim et al., cited above). Recently, however, contrast agents based on iron oxide nanoparticles have been withdrawn from the market, not for safety reasons, but because of too small market share: Gd(III) based contrast agents are preferred for the bright MR images, being Ti relaxing, and they can cover most of the organs including the liver, while those based on iron oxide are used exclusively for the liver (D. Kim et al., cited above). Other contrast agents in the form of nanoparticles are based on manganese oxide (Mn). Mn has a well-defined role in the metabolism of the human body and can be effectively excreted by biological organisms, since the Mn-O bond can be cleaved in reducing or acidic environments showing low toxicity and high biosecurity (G. Yang et al. , cited above). Various manganese oxide (Mhq2) nanostructures have been described as biocompatible for applications as relaxing Ti-type MRI contrast agents (G. Yang et al., cited above).

One of the problems with MRI is the need to overcome inherent limitations such as low sensitivity and artefacts. For example, hyperintense or hypointense signals can be generated by endogenous factors such as fat, air, bleeding, calcification or metal deposits, which are sometimes confused with MRI signals generated by contrast media (D. Kim et al.). Indeed, although much information can be gathered from a single medical imaging modality, various imaging techniques are required to provide comprehensive quantitative diagnostic information with a high spatial and temporal resolution, high sensitivity, and tomographic capability. For example, magnetic resonance imaging can be used to detect tumors in deep tissues and provide a three-dimensional image of biological structures and processes with sub-millimeter resolution. Instead, the CT scan is useful for discriminating tissues with small differences in their opacity (see W02009032752 on behalf of the inventors).

CT uses differential X-ray attenuation and tissue thickness to create cross-sectional and three-dimensional images. Due to the higher speed, lower cost, improved efficiency and higher spatial resolution for clinical imaging, CT has rapidly replaced film radiography, becoming the most popular imaging tool despite the greater amount of ionizing radiation to which the patient is exposed. (D. Kim et al. and X. Han et al., cited above). At the same time, new advantageous systems and methods are being developed to obtain and reconstruct the tomographies obtained by CT and MRI, based on bidirectional image analysis (see WO2017048856 in the name of Rensselaer Polytech Inst.). The goal is to produce better CT and MRI image quality than would be achieved with individual reconstruction.

CT also uses contrast agents to improve imaging performance. CT contrast agents play a crucial role in discriminating between tissues with similar attenuation coefficients. To date, barium and iodine-based contrast agents have been used in clinical imaging. Since the CT scan can detect a concentration of about 10^-2 M of contrast agent, a high dose must be administered, which implies problems of toxicity and tolerability. For example, although barium sulphate suspension has been administered orally for gastrointestinal imaging for decades, it cannot be used as an intravascular contrast medium due to its renal and cardiovascular toxicity (D. Kim et al., cited above).

Instead, iodine-containing small molecules like iopamidol and iodixanol have been approved as intravenous CT contrast agents by the United States Food and Drug Administration. However, there are still several concerns regarding the safety of iodinated contrast agents, such as allergic reactions or anaphylactic shock, and renal toxicity. Furthermore, the blood circulation time of iodinated contrast agents is very short, preventing their preferential accumulation in the lesions (D. Kim et al., cited above). In addition to toxicity and pharmacokinetics, it should be mentioned that the contrast agents based on barium and iodine do not exhibit an optimal contrast effect at the operating voltage of clinical X-ray tubes (D. Kim et al., cited above). Consequently, nano-sized contrast agents have been introduced to overcome these limitations, often based on Au and Bi (X. Han et al., cited above).

While it is true that it is often necessary to use more than one imaging technique to integrate the strengths of each of them and overcome their limitations to improve diagnostics, preclinical research and therapeutic monitoring, it is also true that each technique uses a different contrast agent. Generally, this means that multiple contrast agents, different for each imaging modality, must be administered to a single subject (see WO2010111066 in the name of Univ. Florida). In this way, the use of more than one bio imaging technique requires additional time and costs and can complicate the diagnostic process. Conversely, having multimodal contrast agents that can be used for more than one imaging technique is highly desirable because the patient would need only one medicine to access various types of imaging. The application of multimodal contrast agents is also particularly important to reduce the cost associated with biological imaging techniques (W02009032752, cited above).

To solve this problem, multimodal contrast agents have been introduced that can assist various techniques such as MRI, CT, PET or US (D. Kim et al., cited above). Often, the problem of inserting multiple functionalities in a single contrast agent has been solved by resorting to nanomaterials.

For example, CN103721271B describes a multifunctional nanotechnology probe composed of a core of gold nanoparticles wrapped with a liposomal layer also containing chelating units for Gd(III) ions.

In CN103223178B under the name of Univ. Donghua a method of preparation of FesCh nanoparticles and gold nanoparticles trapped in a folic acid-modified dendrimer, optimized for bimodal imaging by MRI and CT is described.

In CN102327625B in the name of Ningbo Inst. Mat. Tech & Eng. Cas. a method of preparation of metal (Pt, Ag, Au) and oxide nanoparticles optimized for bimodal imaging by MRI and CT is reported.

In CN104689346B in the name of Xiamen Inst of Rare Earth Materials, a multifunctional nanoprobe of Au, PPy and FesCh that can be applied to MRI, CT and guided photothermal therapy is described. The patient can simultaneously obtain diagnosis and treatment by a single administration of reagent.

The CN105641696 in the name of Nat. Ct Nanoscience & Technology China reads about an invention involving a gold-gadolinium nano-composite material comprising an inner core of a silicon dioxide-modified gold nano-rod and a gadolinium-containing silicon dioxide layer covering the core. The material has useful properties for MRI, CT, as well as photo-acoustic imaging and photothermal therapy.

In W02015070036 in the name of Univ. Johns Hopkins, magnetic iron oxide (MION) nanoparticles with silica (SiMION) and gold-silica nanospheres (AuSiMION) are claimed, for MRI, CT scan as well as magnetic hyperthermia and optical imaging.

In WO2010111066 in the name of Gen Hospital Corp. the problem of chemically linking several entities together is considered, describing the preparation of iron oxide nanoparticles coated with dextran, on which gold nanoparticles are conjugated. The nanosystem can be used for MRI and CT.

EP2043700 in the name of Boston Scient. Ltd. describes the coating of biomedical implants with radiopaque or MRI nanoparticles, which disassemble and can be eliminated from the body over time, with the function of facilitating the location of the implant and its positioning only for the time necessary.

In some of the above examples, the contrast agents have a core-shell structure. However, to date, there have been significant problems in developing these core-shell structures for clinical applications, as some of the currently available particles require the use of toxic chemicals during synthesis, which limit the use of the contrast agent in the human body, or possess a morphology that prevents the particles from being effectively functionalized. Other types of multimodal contrast agents include multiple monomodal entities that support a distinct bioimaging modality. Multiple entities are typically joined together using chemical linkers to create single particles, each containing all types of monomodal entities. However, chemical ligands often have different stability in cells and tissues or over time, which means that some entities could separate, thus degrading the quality and usefulness of these contrast agents. Furthermore, different materials have different reactivity with chemical ligands, which forces to long and difficult procedures of surface conjugation in the pharmaceutical field at an industrial level.

In the aforementioned cases in which systems based on Fe and Au are described, these are not-degradable structures in the Au component, and in any case not constituted by alloys of Au and Fe obtainable in a single phase, but require several synthesis steps and the assembly of different components.

An exception is a work reported in the literature (V. Amendola et al., Magneto-Plasmonic Au-Fe Alloy Nanoparticles Designed for Multimodal SERS-MRI-CT Imaging, Small (2014), vol 12, pages 2476-2486), where nanoparticles of Au and Fe alloys are obtained as a single phase, in a single step. However, these nanoparticles are not degradable, as explicitly reported in the above cited paper.

In W0 2004/108165 A2 in the name of Consejo Superiorlnvestigacion, ultrasmall (less than 2.5 nm) of Au, Au/Fe, Au/ Cu, Au/ Gd, Au/Fe/ Cu, Au/Fe/ Gd or Au/Fe/ Cu/ Gd nanoparticles with potential application in imaging or therapy are described. These nanoparticles are already ultrasmall and there is no information on the composition required to obtain biodegradable alloys that can change their size over time from tens of nanometers (for optimal biodistribution in vivo) to few nanometers (for clearance) and are also efficient for imaging or therapy purposes. The claimed ratio of Au to the other elements is in the 5:01 to 5:1 range, where no biodegradability was observed in biological environments (Small (2014), vol 12, pages 2476-2486, cited above).

US 2014/ 0194280 in the name of Osaki Mayuko et Al. and Nanoscale, 2018, 10, 16334 reported the synthesis of Au-Fe nanoparticles by, respectively, chemical reduction or by laser ablation. These studies do not demonstrate the exploitability of the Au-Fe alloy nanoparticles for multimodal imaging and/ or therapy and do not provide any indication about how to achieve biodegradable alloy nanoparticles that are also efficient for imaging or therapy purposes. In Nanoscale, 2018, 10, 16334, the formation of inhomogeneous core- shell structures instead of homogeneous alloys is also described.

Where imaging must be followed by therapy in the analyzed area , it is extremely advantageous to have a contrast agent that can also act as an adjuvant to therapy, in particular for the most commonly used therapies.

Radiotherapy based on external radiation is a widely used cancer treatment strategy in the clinic, being applied to the treatment of 65-75% of solid tumors at various stages (Q. Chen et al., cited above). During radiation therapy with X-rays (XRT), beams of ionizing radiation such as high-energy X-rays, g-rays, or electron beams are applied topically to tumors to kill cancerous cells. Radiation therapy is a powerful type of treatment where precision and accuracy are paramount." Image-guided radiotherapy" (IGRT) facilitates accurate verification of the position of the target tissue, considering anatomical changes related to internal organ movement. The IGRT thus helps reduce the toxicity of radiotherapy and relapse.

Given the possibility of including multiple functionalities in nanomaterials, the combination of diagnostic and radiosensitization functions in the same nanoparticle has been proposed several times.

For example, in W0200637081 and US2008003183 under the name of Univ. California it is described the nanoparticle enhanced X-ray therapy (NEXT), which uses nanomaterials as radiosensitizers to improve the absorption of electromagnetic radiation in specific cells or tissues. Radiosensitizing nanomaterials emit Auger electrons and generate radicals in response to electromagnetic radiation, which can cause localized damage to DNA or other cellular structures such as membranes. Radiosensitizing nanomaterials can also be used as detection agents to aid in the early diagnosis of the disease, along with well- established techniques such as CT.

In WO2018215595 in the name of Technical Univ. Of Denmark And Nanovi Radiotherapy Aps, reference is made to the fact that, currently, the most frequently used imaging technique for IGRT is CT. However, CT-based target delineation of soft tissue tumors tends to improve the precision but not the accuracy of treatment due to the relatively low resolution for soft tissue. In some tumors, about 40% of the volume is estimated by CT scan over MRI, which has sub-millimeter resolution for soft tissue. Therefore, it is advantageous to apply both CT and MRI in the planning of radiotherapy of soft tissue tumors. To solve this problem, WO2018215595 describes a marker based on complexes of Gd(III) and XSAIB (a CT contrast agent in sucrose), useful for radiotherapy guided by MRI and CT. In EP2593186 for Univ. Denmark Tech Dtu, nanometer-sized particles are optimized for CT image-guided radiotherapy of the target tissue. More specifically, the invention relates to nanoparticles composed of elements which act as solid-form contrast agents for X-ray imaging, enabling simultaneous or integrated external beam radiotherapy with computed tomography.

In CN108514642 to Univ. National Dong Hwa Shanghai 10th Peoples Hospital, FesCh and Au nanoparticles are described as having excellent biocompatibility and good performance for imaging by MRI, CT and PA. Nanoparticles can be used for photothermal cancer therapy, enhanced through radiotherapy.

US9121049 in the name of the authors relates to a pharmaceutical composition for increasing the radiation sensitivity of cancer cells, which includes: a nanoparticle containing a first element, which is Pt, and a second element which is iron, cobalt, palladium, silver, nickel, copper or gadolinium. The nanoparticle is a metal nanoparticle, an alloy nanoparticle or a metal nanoparticle with a core-shell structure, and the size of the nanoparticle is under a controllable range of 3 nm to 150 nm. Furthermore, the present invention provides a detection method through CT or MRI. Furthermore, the present invention provides a pharmaceutical composition for elevating the radiation sensitivity of cancer cells, to improve the efficiency of radiation therapy for cancer cells.

Overall, the aforementioned multimodal agents for MRI and CT and sensitizers for X-ray radiotherapy frequently contain heavy or toxic metals, especially Gd(III). In cases where systems based on Fe and Au are described, these are non-degradable structures in the Au component, and in any case not made of alloys of Au and Fe obtainable in a single phase, but require several synthesis steps and assembly of different components.

Neutron capture therapy (NCT) is another type of radiotherapy in which there is a pressing need to locate and quantify the sensitizer to calibrate the neutron dose to be used for patient treatment. Boron Neutron Capture Therapy (BNCT) is a radiotherapy treatment modality based on the accumulation of a drug containing the ¹⁰B isotope and subsequent irradiation with low-energy neutrons, inducing the decay of ¹⁰B in ⁷Fi and a a particle. The high FET (linear energy transfer) of the two particles ⁷Fi and a causes the death of the neoplastic cells that have accumulated B atoms. A variant consists of GdNCT, which however is associated with the generation of Auger electrons which have FET considerably lower, despite the neutron capture cross-section of the ¹⁵⁷Gd isotope is higher than that of ¹⁰B.

The need to accumulate high amounts of radiosensitizer in the tissue to be treated and to quantify the accumulation using tomographic imaging techniques has led to the development of several multifunctional systems for NCT, mainly molecular but also in the form of nanomaterials.

For example, EP2836237 in the name of Universite Claude Bernard Lyon and Nano H relates to ultrafine nanoparticles useful as a diagnostic and therapeutic agent, which can be administered through the airways. The nanoparticles consist of a silica matrix containing various elements useful for MRI, CT, radiosensitization and NCT, and based on lanthanides and Gd.

In W02009032752 already mentioned above, a silicon oxide-based nanosystem is described which incorporates chelating functions for Gd ions, the whole surface covered with a layer of gold. The system can be used for MRI and GdNCT.

In EP2099304 and US8287839 in the name of Brookhaven Sciences Ass Lie, low toxicity boron compounds are considered for the diagnosis and treatment of tumors. More specifically, the invention consists of porphyrin compounds containing carborane with halide, amine or nitro groups and methods for their use in particular in boron neutron capture therapy (BNCT), XRT radiotherapy and photodynamic therapy (PDT) for the treatment of tumors of the brain, head and neck and surrounding tissues. The invention is also directed at using these carboran-containing porphyrin compounds in imaging and / or tumor diagnosis methods such as MRI, SPECT or PET. The metal can be vanadium, manganese, iron, ruthenium, technetium, chromium, platinum, cobalt, nickel, copper, zinc, germanium, indium, tin, yttrium, gold, barium, tungsten or gadolinium.

In EP2200659 in the name of Univ Claude Bernard Lyon and Hospices Civils Lyon, nanoparticles having dimensions between 1 and 50 nm are considered, consisting of an oxide or a hydroxide of at least one lanthanide, or a nucleus of hydroxide of at least one lanthanide and a coating consisting of a polysiloxane, optionally with organic molecules grafted to the surface or embedded therein. Such nanoparticles work as injectable radiosensitizing agents aimed at improving the efficacy of treating a tumor by X-ray or gamma irradiation or gadolinium neutron capture therapy.

In US2007093463 in the name of Brookhaven Science Ass. Lie a low toxicity carborane- containing tetraphenylporphyrin compound is claimed, for use in particular in boron neutron capture therapy (BNCT), XRT radiotherapy and photodynamic therapy (PDT). The invention also includes tumor imaging and / or diagnosis methods such as MRI, SPECT or PET using these halogenated compounds containing tetraphenylporphyrin and carborane. In US8557290 in the name of Univ. Northwestern, iron oxide nanoparticles are described with an outer layer of titanium oxide, on the surface of which other elements such as Gd are coordinated, usable for MRI, CT, GdNCT, in addition to other therapeutic applications.

Overall, the aforementioned multifunctional agents frequently contain heavy or toxic metals, in particular Gd(III). Only in rare cases, the invention is compatible simultaneously for different imaging and therapy modalities such as MRI, CT, XRT and BNCT. In cases where systems based on Fe, Mn, B or Au are described, these are non- degradable structures in the Au component, and in any case not made up of alloys obtainable in a single phase, but require several synthesis steps and the assembly of different components.

In the aforementioned patents, the synthesis methods used to obtain multifunctional organic nanomedicines consist in the realization of polymeric and / or protein aggregates on which the specific functions required for multimodality are present, such as, for example, functional groups able to stably coordinate metal ions, hooked to the main molecular scaffold through suitably developed organic synthesis procedures.

The synthesis methods used to obtain multifunctional inorganic nanomedicines consist in reducing metal salts in suitable conditions of temperature, pressure and solvation environment, to direct the assembly of the different chemical elements towards the formation of ordered structures with very precise geometry and dimension. Subsequently or simultaneously with the formation of these inorganic nanostructures, they are coated with a layer of polymers or other biocompatible substances aimed at guaranteeing colloidal stability in biological fluids.

In none of the above cases reference is made to synthesis techniques that do not require toxic and / or expensive chemicals, and which act in conditions far from thermodynamic equilibrium, for the preparation of multifunctional nanoparticles. In fact, the above cases never consist of disordered or metastable phases (i.e. kinetically stable but not thermodynamically stable).

Many of the systems described in the state of the art have a molecular structure and, in addition to containing Gd, have the drawbacks of biodistribution and immediate renal accumulation of molecular compounds. The coordination of Gd(III) in many compounds, including porphyrin derivatives, is characterized by a significantly lower association constant than that of the most efficient chelators for MRI such as DOTA, and the release of Gd by cation exchange with any high concentration metal ion present in the organism (in particular Fe(III)) would be immediate.

In general, the biocompatibility of inorganic nanoparticles is a critical factor (D. Kim et al., cited above). There are currently about 50 nanopharmaceuticals that have been approved by the Food and Drug Administration (FDA), and the main source of delay for translation into clinical practice relates to biodistribution and safety of nanoparticles (X. Han et al., cited above). It takes an average of 12 years for a new drug to move from invention to clinical application with FDA approval, and safety remains a critical aspect of this process (X. Han et al., cited above).

On the other hand, modification of nanoparticles to increase their circulation time is crucial for imaging and therapy performance. The most common modification method is the encapsulation of hydrophobic nanomaterials in a polyethylene glycol (PEG) shell, which greatly increases the solubility and prolongs the blood circulation time. Due to the hydrophilic structure of the PEG, the binding of opsonins to nanoparticles and the recognition by macrophages is significantly lower, reducing the sequestration of nanoparticles by the reticuloendothelial system (RES), and in particular by the liver, spleen and lungs, thus prolonging the half-life of blood circulation (G. Yang et al. and X. Han et al., cited above). For passive targeting, the circulation time and the size of the nanoparticle, which can range from several nanometers to hundreds of nanometers, are the most important characteristics (X. Han et al., cited above).

Therefore, inorganic nanoparticles with multiple and diversified properties, despite having enormous potential in the field of nanomedicine, must overcome the problems of toxicity in healthy tissues and organs, which has led to their limited transposition in the clinic to date. This problem can be limited by the emergence of degradable inorganic nanoparticles or nanoparticles with dimensions compatible with the accumulation in the organs deputed to the removal of foreign bodies from the organism (G. Yang et al.).

In summary, the problems typically encountered in the conception, realization and application of multifunctional medicines or nanomedicines for diagnostic techniques such as MRI or CT scan and for radiotherapy with X-rays or neutrons consist of one or more of the following cases:

- presence of non-biocompatible or toxic substances such as rare earths, heavy metals, toxic metals;

- limited colloidal stability, resulting in limited accumulation in the tissues to be treated, in association with an immediate and massive accumulation in the systems or organs responsible for the removal of foreign bodies from the organism; - rapid renal removal of compounds with reduced dimensions (typical of molecular compounds), with a consequently limited accumulation in the tissues to be treated, except for massive administration of the substance, with consequent considerable accumulation in the kidney. This also entails significantly long infusion / administration times;

- adverse reactions due to hepatotoxicity, accumulation in organs of the nervous system, allergic reactions, excessively long infusion times. For example, gadolinium-based contrast agents increase the risk of nephrogenic systemic fibrosis (NSF) in patients with acute or chronic severe renal insufficiency (glomerular filtration rate less than 30 mL/ min / 1.73 m²) or acute renal failure of any severity due to hepatorenal syndrome or in the perioperative hepatic transplant period;

- bio-persistence of the substance in the body for an indefinite time, due to its chemically inert nature and large size;

- impossibility of synthesizing the multifunctional agents due to thermodynamic limitations that prevent their formation under conventional synthesis conditions (starting from metal salts);

- expensive synthesis procedure, not scalable on quantities necessary for clinical use, or not compatible with the reproducibility, sterility and safety procedures required for clinical use;

- poor efficacy due to the low density of functional elements for diagnostic or therapeutic purposes, often accentuated by the limited permeation of the substance in the area of interest, at the level of tissue or cellular penetration.

All the above problems are multiplied by the number of different substances to be used when it is necessary to administer different monofunctional contrast or therapeutic agents. Besides, one should consider also the limits related to the use of monofunctional substances, stated previously, including for example the impossibility of dosing and localizing the sensitizer, evaluating the efficacy of administration and calibrating the dosage and spatial distribution of radiation for therapy.

In order to solve many of the aforesaid problems, and to obtain the advantages highlighted below, the Proponents have studied, tested and implemented the present invention.

DEFINITIONS

Unless otherwise defined, all the terms of the art, notations and other scientific terms used here are intended to have the meanings commonly understood by those who are experts in the technique to which this description belongs. In some cases, terms with commonly understood meanings are defined here for clarity and / or for ready reference; the inclusion of these definitions in this description should therefore not be interpreted as representing a substantial difference from what is generally understood in the art.

The terms "treat", "treating" and "treatment" refer to a method of alleviating or eliminating a disease and / or its associated symptoms.

The terms "prevent", "preventing" and "prevention" refer to a method of preventing the onset of a disease and / or its associated symptoms or preventing a person from acquiring a disease. As used herein, "prevent", "preventing" and "prevention" also include delaying the onset of a disease and / or its associated symptoms and reducing a person's risk of contracting a disease. The term "therapeutically effective amount" refers to that amount of the compound to be administered sufficient to prevent the development or to some degree relieve one or more of the symptoms of the condition or disorder being treated.

The terms "comprising", "having", "including" and "containing" are to be understood as open terms (i.e. the meaning "comprising, but not limited to") and are to be considered as support also for terms such as "essentially consist of", "consisting essentially of", "consist of " or "consisting of".

The terms "essentially consists of", "essentially consisting of" are to be understood as semi-closed terms, which means that no other ingredients that affect the new characteristics of the invention are included (optional excipients can therefore be included).

The terms "consists of", "consisting of" are to be understood as closed terms.

The acronym "MRI" refers to the instrumental technique of nuclear magnetic resonance imaging.

The acronym "CT" refers to the instrumental technique of X-ray computed axial tomography. The acronym "XRT" refers to the instrumental technique of radiotherapy using X-rays.

The acronym "NCT" refers to the instrumental technique of radiotherapy using neutron capture.

The acronym "BNCT" refers to the instrumental technique of neutron capture radiotherapy with boron.

With the acronym "NM" we mean "nanomaterial".

With the acronym "NMs" we mean "nanomaterials".

The acronym "PET" refers to the instrumental technique of positron emission tomography. The acronym "US" refers to the term ultrasound, unless the acronym US is placed immediately before numbers, and in this case it indicates the numbers of US patent documents instead.

The terms "imaging" or "bio-imaging" mean the visualization of biological structures and processes. The acronym "IGRT" refers to image-guided radiotherapy.

The acronym "NEXT" refers to X-ray therapy enhanced with nanoparticles.

With the acronym "LET" we mean linear energy transfer.

The acronym "SPECT" refers to single-photon emission tomography.

The acronym "PDT" refers to photodyna ic therapy. The acronym "DOTA" refers to the dodecanotetraacetic acid chelator.

The acronym "RES" refers to the reticuloendothelial system.

The acronym "NSF" refers to nephrogenic systemic fibrosis.

The term "geometric dimension" means the dimension measured with the transmission electron microscope. The terms "EDX analysis" means the "EDX" spectroscopic analysis from the English energy dispersive X-ray analysis. The acronym "PEG" refers to polyethylene glycol.

The acronym "FCS" refers to foetal calf serum.

The acronym "PBS" refers to the phosphate-buffered saline.

The acronym "DLS" refers to the dynamic light scattering.

The acronym "FTIR" refers to Fourier transform IR spectroscopy.

The acronym "TEM" refers to transmission electron microscopy.

With the acronym "F.C.C." we mean a face-centered cubic system.

The symbols Gd, Au, Fe, Mn, B, Ag, O, C etc etc are the symbols of the chemical elements as reported in the periodic table of elements.

With the expression "AuFe NPs" or with the expression "Au-Fe NPs" we mean nanoparticles of an alloy of gold and iron, also called Au-Fe nanoalloys or alloy nanoparticles.

With the expression "FeB NPs" or with the expression "Fe-B NPs" we mean nanoparticles of an alloy of iron and boron, also called Fe-B nanoalloys or alloy nanoparticles.

With the expression "AuFeB NPs" or with the expression "Au-Fe-B NPs" we mean nanoparticles of an alloy of gold, iron and boron, also called Au-Fe-B nanoalloys or alloy nanoparticles.

The abbreviation "HU" refers to the Hounsfield (HU) units for measuring radiodensity.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the drawbacks of the known art.

In particular, the object of the present invention are nanoparticles comprising a metallic nanoalloy and a biocompatible organic stabilizer, useful in the diagnosis and therapy of various pathological conditions, including cancer.

These and other objects of the present invention are achieved using a text incorporating the characteristics of the attached claims, which form an integral part of the present description.

In one embodiment, the invention relates to nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprising a metal nanoalloy and a biocompatible organic stabilizer in which: a) the nanoalloy essentially consists of X-Y-Z, in which X, Y and Z are different from each other and in which X can be gold and / or silver or iron and / or manganese, Y can be iron, manganese or boron and Z can be silver, manganese, boron or may be absent; and b) when Z is present, X varies in an atomic percentage range between 5% and 95%, Y varies in an atomic percentage range between 2.5% and 85% and Z varies in an atomic percentage range between 2.5% and 50%; when Z is absent, X and Y vary in an atomic percentage range between 5% and 95% proviso that when X is gold and Y is iron, X is present in atomic percentage between 15% and 80% and Y is present in atomic percentage between 20% and 85%.

A different aspect of the present invention also refers to the combined, simultaneous and / or spatially coincident use of the nanoparticles of the invention as contrast agents in diagnostic techniques based on the acquisition of images of the human and / or animal body, such as for example for MRI and / or CT, and as sensitizing agents in radiotherapy techniques such as XRT and / or NCT.

Further characteristics and objects of the present invention will become clearer from the following description.

ILLUSTRATION OF THE DRAWINGS

The invention will be described below with reference to some examples, provided for explanatory and non-limiting purposes, and illustrated in the attached drawings. These drawings illustrate different aspects and embodiments of the present invention and, where appropriate, reference numbers illustrating structures, components, materials and / or similar elements in different figures are indicated by similar reference numbers.

Figure 1 shows a scheme for the production of nanoparticles by laser ablation in liquid: the pulses emitted by a laser (a) are deflected through a system of optics (b), which can be fixed or mechanized, and focused through suitable lenses (c) inside a cell (d) containing the liquid and the metal target (e). The liquid inside the cell can remain for the duration of the experiment, or it can be changed using a pump (f) that draws from a reserve (g) and discharges into a container (h).

Figure 2 relates to the structural characterization of Au-Fe nanoparticles: it shows the results of the semiquantitative EDX analysis performed on Au-Fe nanoparticle powder, demonstrating the presence of Au and Fe, as well as C and O (the latter are typical of the PEG coating).

Figure 3 relates to the structural characterization of Au-Fe alloy nanoparticles: it shows the size distribution measured by DLS for a solution of Au-Fe alloy nanoparticles in FCS for 24 h.

Figure 4 relates to the structural characterization of Au-Fe nanoparticles: it shows the results of the FTIR analysis performed on Au-Fe nanoparticle powders, which exhibits the characteristic absorption peaks of the polyethylene glycol that covers the nanoparticles (region 800 - 1500 cm ¹ and 2800 cm⁴).

Figure 5 relates to the structural characterization of Au-Fe nanoparticles: it shows TEM images of Au-Fe nanoparticles mixed with FCS at lysosomal pH (pH = 4.5) for a time equal to 0 h, 2 weeks and 2 months. After 0 h, the nanoparticles have a spherical shape and sizes ranging from 1 to 60 nm. After 2 weeks, the nanoparticles have a crescent or doughnut shape, and sizes between 1 and 25 nm. After 2 months, the nanoparticles have a spherical shape and dimensions between 1 and <10 nm.

Figure 6 relates to the structural characterization of Au-Fe nanoparticles: it shows the size distribution measured by TEM (geometric dimension), on Au-Fe nanoparticles mixed with FCS at lysosomal pH (pH = 4.5) for 0 h (squares), 2 weeks (triangles), and 2 months (circles). The progressive decrease in the size of the nanoparticles with time is observed.

Figure 7 relates to the structural characterization of Au-Fe nanoparticles: it shows the results of the XRD analysis performed on a powder sample of Au-Fe nanoparticles, which exhibits the reflections of the F.C.C. lattice of Au, but at different angles corresponding to the formation of an Au-Fe alloy where the Fe atoms occupy the sites of the crystal lattice according to a random distribution.

Figure 8 refers to the functional characterization of Au-Fe alloy nanoparticles: it shows T2-weighted MRI images of phantoms containing decreasing amounts of Au-Fe alloy nanoparticles as reported in the table. The Au-Fe nanoparticles generate a negative contrast with an entity related to the concentration of the nanoparticles in the phantoms.

Figure 9 refers to the functional characterization of Au-Fe alloy nanoparticles: the graph of the relaxivity ¾ as a function of the Fe concentration, corresponding to an average relaxivity in the linear zone of 22 mM(Fe) ¹ s ¹ is shown.

Figure 10 refers to the functional characterization of Au-Fe alloy nanoparticles: it shows CT images of phantoms containing decreasing quantities of Au-Fe alloy nanoparticles, as reported in the table. It is observed that the Au-Fe alloy nanoparticles generate a positive contrast with an entity correlated to the concentration of the nanoparticles in the phantoms.

Figure 11 refers to the functional characterization of nanoparticles in Au-Fe nanoalloys: it shows the X-ray attenuation graph (measured in HU) as a function of the Au concentration in the tubes. The result agrees with what is expected for Au-based compounds.

Figure 12 shows CT scans of the spleen taken before and after injection of Au-Fe nanoparticles into the tail vein of healthy mice, and mean HU values at different times.

Figure 13 shows liver CT images acquired before and after the injection of Au-Fe nanoparticles into the tail vein of healthy mice, and mean HU values at different times.

Figure 14 reports MRI images acquired before and after the injection of Au-Fe nanoparticles into the caudal vein of healthy mice, and values of the T2 signal decrease (mean) at different times and in different regions of interest (liver and spleen).

Figure 15 refers to mean HU values measured in mice tumor models as a function of time after administration of Au-Fe nanoparticles, of reference pure Au nanoparticles and of the control (no nanoparticles administered).

Figure 16 refers to the structural characterization of Fe-B alloy nanoparticles: it shows the semi-quantitative EDX analysis performed on Fe-B alloy nanoparticle powder, indicating the presence of Fe and B, as well as C and O (specific to the polyvinylpyrrolidone coating).

Figure 17 refers to the structural characterization of Fe-B alloy nanoparticles: it shows the size distribution measured by DLS for a solution of Fe-B alloy nanoparticles in FCS, PBS and water.

Figure 18 refers to the structural characterization of Fe-B alloy nanoparticles: it shows FTIR analysis performed on Fe-B nanoparticle powders, which exhibits the characteristic absorption peaks of the polyvinylpyrrolidone that covers the nanoparticles (region 1100 - 1700 cm ¹ and 800 - 600 cm⁴).

Figure 19 refers to the structural characterization of Fe-B alloy nanoparticles: TEM images of Fe-B nanoparticles mixed with FCS at lysosomal pH (pH = 4.5) for a time equal to 0 h and 1 month are shown. After 0 h, the nanoparticles have a spherical shape and sizes ranging from 1 to 90 nm. After 1 month, the nanoparticles still have a spherical shape but the dimensions are between 1 and <20 nm.

Figure 20 refers to the structural characterization of Fe-B alloy nanoparticles: it shows the size distribution measured by TEM (geometric dimension), on Fe-B alloy nanoparticles mixed with FCS at lysosomal pH (pH = 4.5) for 0 h (squares) and 1 month (circles). The progressive decrease in the size of the nanoparticles with time is observed.

Figure 21 refers to the structural characterization of Fe-B alloy nanoparticles: it shows the XRD analysis performed on a powder sample of Fe-B alloy nanoparticles, which exhibits the characteristic reflections of orthorhombic (Pnma) and cubic (B.C.C.) cells of Fe-B, in addition to a conspicuous background attributed to the presence of an amorphous component.

Figure 22 refers to the functional characterization of Fe-B alloy nanoparticles: it shows T2- weighted MRI images of phantoms containing decreasing quantities of Fe-B alloy nanoparticles, as reported in the table. The Fe-B nanoparticles generate a negative contrast with an entity related to the concentration of the nanoparticles in the phantoms.

Figure 23 refers to the functional characterization of Fe-B alloy nanoparticles: it shows the graph of the relaxivity ¾ as a function of the Fe concentration, corresponding to an average relaxivity in the linear zone of 82 mM(Fe) ¹ s ¹.

Figure 24 refers to the functional characterization of Fe-B alloy nanoparticles: it shows the graph of the intensity of a particles generated during a neutron activation autoradiography experiment, reported as a function of the number of B atoms (isotope ¹⁰B) deposited. The result of the Fe-B alloy nanoparticles is comparable or superior to that of a reference boron compound consisting of H₃BO₃.

Figure 25 shows T2-weighted MRI images acquired before and after the injection of Fe-B alloy nanoparticles into the caudal vein of healthy mice.

Figure 26 shows T2-weighted signal decrease (mean) values at different times after injection of Fe-B alloy nanoparticles into the caudal vein of healthy mice.

Figure 27 relates to the structural characterization of Au-Fe-B alloy nanoparticles: it shows the semiquantitative EDX analysis performed on Au-Fe-B alloy nanoparticle powder, indicating the presence of Au, Fe and B, as well as C and O (typical of the PEG coating).

Figure 28 relates to the structural characterization of Au-Fe-B alloy nanoparticles: it shows the size distribution measured by DLS for a solution of Au-Fe-B alloy nanoparticles in FCS since 24 h.

Figure relates to the structural characterization of Au-Fe-B alloy nanoparticles: it shows the FTIR analysis performed on Au-Fe-B alloy nanoparticle powders, which exhibits the absorption peaks characteristic of the polyethylene glycol covering the nanoparticles (region 800 - 1500 cm ¹ and 2800 cm⁴).

Figure 30 relates to the structural characterization of Au-Fe-B alloy nanoparticles: it shows TEM images of Au-Fe-B alloy nanoparticles mixed with FCS at lysosomal pH (pH = 4.5) for a time equal to 0 h, 1 month and 2 months. After 0 h, the nanoparticles have a spherical shape and dimensions between 1 and 40 nm. After 1 month, the nanoparticles have a spherical shape but dimensions between 1 and 25 nm and show evident signs of corrosion. After 2 months, the nanoparticles have a spherical shape and size between 1 and 10 nm.

Figure 31 relates to the structural characterization of Au-Fe-B alloy nanoparticles: it shows the size distribution measured by TEM (geometric dimension), on Au-Fe-B alloy nanoparticles mixed with FCS at lysosomal pH (pH = 4.5) for 0 h (squares), 1 month (circles) and 2 months (empty squares). The progressive decrease in the size of the nanoparticles with time in the 10-40 nm range is observed.

Figure 32 relates to the structural characterization of Au-Fe-B alloy nanoparticles: it shows the XRD analysis performed on a powder sample of Au-Fe-B alloy nanoparticles, which exhibits the reflections characteristic of F.C.C. cell.

Figure 33 relates to the functional characterization of Au-Fe-B alloy nanoparticles: it shows T2-weighted MRI images of phantoms containing decreasing amounts of Au-Fe-B alloy nanoparticles, as reported in the table. It is observed that the Au-Fe-B nanoparticles generate a negative contrast with an entity correlated to the concentration of the nanoparticles in the phantoms.

Figure 34relates to the functional characterization of Au-Fe-B alloy nanoparticles: it shows the graph of the relaxivity ¾ as a function of the Fe concentration, corresponding to an average relaxivity in the linear zone of 59 mM(Fe) ¹ s ¹.

Figure 35 relates to the functional characterization of Au-Fe-B alloy nanoparticles: it reports CT images of phantoms containing decreasing amounts of Au-Fe-B alloy nanoparticles, as reported in the table. It is observed that the Au-Fe-B alloy nanoparticles generate a positive contrast with an entity correlated to the concentration of the nanoparticles in the phantoms.

Figure 36 relates to the functional characterization of Au-Fe-B alloy nanoparticles: it shows the X-ray attenuation graph (measured in HU) as a function of the concentration of Au in the tubes. The result agrees with what is expected for Au-based compounds.

Figure 37 relates to the functional characterization of Au-Fe-B nanoparticles: it shows the graph of the intensity of a particles generated during a neutron activation autoradiography experiment, reported as a function of the number of atoms of B (isotope ¹⁰B) deposited. The result of the Au-Fe-B alloy nanoparticles is comparable or superior to that of a reference boron compound consisting of H3BO3.

DETAILED DESCRIPTION OF THE INVENTION

While the invention is susceptible to various alternative modifications, some preferred embodiments are described below in detail. It should be understood, however, that there is no intention of limiting the invention to the specific embodiment illustrated, but, on the contrary, the invention is intended to cover all modifications, alternatives, and equivalents that fall within the scope of the invention, as defined in the claims.

The use of "for example", "etc", "or" indicates non-exclusive alternatives without limitation unless otherwise indicated. The use of "include" means "includes, but not limited to" unless otherwise indicated.

The present invention relates to nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprising a metal nanoalloy and a biocompatible organic stabilizer in which: a) the nanoalloy essentially consists of X-Y-Z, in which X, Y and Z are different from each other and in which X can be gold and / or silver or iron and / or manganese, Y can be iron, manganese or boron and Z can be silver, manganese, boron or may be absent; and b) when Z is present, X varies in an atomic percentage range between 5% and 95%, Y varies in an atomic percentage range between 2.5% and 85% and Z varies in an atomic percentage range between 2.5% and 50%; when Z is absent, X and Y vary in an atomic percentage range between 5% and 95% with proviso that that when X is gold and Y is iron, X is present in atomic percentage between 15% and 80% and Y is present in atomic percentage between 20% and 85%.

According to a preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is gold, Y is iron and Z is absent; and b) X is present in an atomic percentage between 15% and 80% and Y is present in an atomic percentage between 20% and 85%.

According to a preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is gold, Y is manganese and Z is absent; and b) X and Y are each present in atomic percentages between 10% and 90%.

According to another preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is silver, Y is iron or manganese and Z is absent; and b) X and Y are each present in atomic percentages between 10% and 90%.

According to another preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is iron and / or manganese, Y is boron and Z is absent; and b) X is present in an atomic percentage between 20% and 95% and Y is present in an atomic percentage between 5% and 80%.

According to a particularly preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is iron, Y is boron and Z is absent; and b) X is present in an atomic percentage equal to 75% and Y is present in an atomic percentage equal to 25%.

According to a preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is gold and / or silver, Y is iron and / or manganese and Z is boron; and b) X is present in atomic percentage between 10% and 55%, Y is present in atomic percentage between 20% and 85% and Z is present in atomic percentage between 5% and 50%.

According to another particularly preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is gold, Y is iron and Z is boron; and b) X is present in an atomic percentage equal to 52%, Y is present in an atomic percentage equal to 22% and Z is present in an atomic percentage equal to 26%.

According to a further particularly preferred aspect, the nanoparticles with a geometric dimension between 1 and 1000 nm, preferably between 1 and 500 nm, comprise a metal nanoalloy and a biocompatible organic stabilizer in which: a) X is gold, Y is iron and Z is absent; and b) X and Y are each present in an atomic percentage equal to 50%.

In the nanoparticles of the invention, the structure of the metal nanoalloy can be ordered crystalline, disordered crystalline, defective crystalline and / or amorphous, preferably it is a mixed crystalline / amorphous or disordered crystalline structure, as indicated by transmission electron microscope (TEM) images at high resolution (Fig. 5, 6, 19, 20, 30 and 31) and by X-ray diffraction (XRD) analysis (Fig. 7, 21 and 32).

Where the dimensions of the nanoparticles exceed the optimal threshold for effective removal from the organism, the chemical composition and the partly crystalline and partly amorphous structure make it possible to degrade the nanoparticles in times of the order of days or weeks, as indicated by the TEM and XRD analysis.

The nanoparticles of the invention are characterized by being degradable in a biological fluid, for example they are degradable in a liquid solution containing biological or bioproduced components, such as for example human or bovine serum, blood or any other fluid present in a living organ, characterized by physiological pH (at a pH of about 7-8) or acid / lysosomal (at a pH of about 4.5). Bovine serum can be used as an equivalent to human serum or human blood.

Preferably, the nanoparticles of the invention are degradable in serum at a pH between 4 and 5.

After degradation in a biological fluid, the geometric dimension of the nanoparticles of the invention is between 1 and 60 nm, preferably between 1 and 15 nm.

To give colloidal stability to the particles in biological fluids, the nanoparticles of the invention are coated with a biocompatible organic stabilizer.

Biocompatible organic stabilizers are for example biocompatible polymers with a molecular weight between 100 Da and 300,000 Da, preferably selected from polyethylene glycol, thiolated polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, cellulose, dextran and / or pullulan.

Other biocompatible organic stabilizers useful for coating the particles of the invention may be thiolated molecules, preferably selected from glutathione, mercaptopropionic acid, mercaptopropansulfonate and / or mercaptophenylboronic acid.

The particular chemical composition and crystalline structure of the alloy nanoparticles of the invention can be obtained with rapid condensation processes of metal elements in nanophases, in the presence of oxygen or in an atmosphere of inert gases to limit oxidation of the elements themselves.

Preferably, in fact, they are obtainable through the laser ablation process or the laser spray pyrolysis process.

This is essential to circumvent the thermodynamic limitations to the coexistence of the different elements such as Au, Ag, Fe, B, Mn in the same metal nanostructure, and allows the assembly of the different elements according to a mixed crystalline / amorphous structure which, in some cases, gives also the degradation properties. Otherwise, the elements that make up the nanoalloy would tend to segregate instead of being homogeneously dispersed on a sub-nanometric scale within each nanoparticle, losing many of the properties that allow its function as multifunctional nanomedicines, i.e. as medicines based on nanoparticles and having multiple diagnostic and / or therapeutic functions at the same time.

In an embodiment of the present invention, the method of synthesis used was that of laser ablation in liquid, which operates according to the scheme shown in Fig. 1: the pulses coming from a laser are focused on a metal target composed of the elements of which the nanoparticles will be made up, and the whole is immersed in a solution of an organic solvent in which an organic stabilizer is dissolved. The pulses emitted by the laser are deflected through an optical system that can be fixed or scanned, and focused through appropriate lenses inside a cell containing the liquid and the metal target to be ablated.

The amount of nanoparticles produced is proportional to the number and energy of the laser pulses arriving on the metal target. The synthesis can be carried out continuously until the metal sheet is consumed, preferably by letting the synthesis-liquid flow into the container through a pump, or by using a container in which a predetermined amount of liquid is present.

This procedure of synthesis is economically sustainable, scalable for the quantities needed for clinical use, and can be made compatible with the reproducibility, sterility and safety procedures required for clinical use.

According to a preferred aspect, the laser ablation process used to obtain the metal nanoparticles of the present invention is performed with laser pulses ranging from 800 nm to 1,200 nm.

According to a further preferred aspect, the laser ablation process used to obtain the metal nanoparticles of the present invention is performed in an organic solvent, preferably in low molecular weight alcohols, ethers and / or ketones, even more preferably in ethanol, isopropanol, tetrahydrofuran and / or acetone, and it is carried out in an ambient atmosphere, or in an inert gas atmosphere, to limit the oxidation of the transition metals present in the nanoalloy.

In a preferred embodiment of the present invention, the metal nanoparticles with a geometric size between 1 and 1000 nm, preferably between 1 and 500 nm comprise a metal nanoalloy where X is Au, Y is Fe and Z is absent, where X is present in an atomic percentage between 15% and 80% and Y is present in an atomic percentage between 20% and 85%, and a biocompatible organic stabilizer.

These nanoparticles are produced through a synthesis method that acts in conditions far from the thermodynamic equilibrium, that is important because the metallic nanoalloy of Au and Fe is not thermodynamically stable at ambient pressure and temperature, except in conditions of high dilution of one of the two elements.

For example, the nanoparticles are obtained by laser ablation in liquid, according to the scheme shown in Fig. 1. The used laser emits pulses with a duration of 1 - 50 ns and at a wavelength of 1064 nm, with an energy per pulse between 10 and 150 mj. It should be noted that other pulsed laser sources, with different characteristics in terms of duration, wavelength and energy per pulse, can also be used for the same purpose.

The solvent used can be ethanol, with volumes ranging from 100 to 500 mL for each individual synthesis. As a biocompatible organic stabilizer, thiolated polyethylene glycol (PEG-SH) is dissolved in ethanol with a concentration between 0.005 and 0.5 mg/ mL.

To obtain these nanoparticles in Au and Fe metal alloy, other organic solvents can also be used, preferably low molecular weight alcohols, ethers and / or ketones, even more preferably ethanol, isopropanol, tetrahydrofuran and / or acetone, both in ambient atmosphere, and in a controlled atmosphere of inert gas, to limit the oxidation of the transition metals present in the nanoalloy.

According to a preferred aspect, the laser ablation process for obtaining the Au and Fe metal nanoparticles is carried out in an inert gas atmosphere.

The thiolated polyethylene glycol has a molecular mass of between 100 and 30,000 Da. Other biocompatible organic stabilizers can also be used to coat the Au and Fe alloy nanoparticles of this preferred aspect of the invention, such as for example but not limited to polyvinylpyrrolidone, cellulose, polyvinyl alcohol, and biocompatible thiolated molecules such as, for example, but not limited to glutathione, mercaptopropionic acid, mercaptopropansulfonate or mercapto phenyl boronic acid.

The biocompatible organic stabilizer is required to obtain colloidal stability in biological fluids and to obtain the complete dispersion of the nanoparticles in the aqueous solution after the removal of the organic solvent.

The metal target used in the laser ablation in liquid synthesis is made of an alloy composed of Au and Fe in a proportion between 80:20 and 10:90 in molar / atomic ratio and obtained through conventional metallurgical techniques.

In this case, in fact, there is a systematic depletion of the iron fraction during the synthesis. The depletion in Fe of the final Au-Fe alloy nanoparticles compared to the composition of the bimetallic target used for synthesis, is due to the oxidation of iron and the formation of non-metallic compounds. These substances are effectively separated from the alloy nanoparticles by the purification and washing procedure described.

The components used and indicated up to now are all known and commercially available. This includes metal foils, which are commercially available or can be self- produced using conventional metallurgical (powder induction heating, powder sintering, electric discharge melting in a controlled atmosphere) or advanced (spark plasma sintering) techniques.

The Au and Fe alloy nanoparticles of this preferred aspect of the invention, preferably obtained by laser ablation in liquid, are collected, stored at room temperature or -20 ° C for the necessary time, then separated from the liquid by centrifugation at speeds ranging from 500 and 30,000 ref for times between 5 and 120 minutes, at temperatures between 20 and -5 ° C. The process is then followed by drying at 20 - 30 ° C, typically using a rotary evaporator at room temperature, or lyophilization of the sediment, and storage in a protective atmosphere at -20 °C or resuspension in the desired aqueous solution, such as phosphate-buffered saline (PBS), assisted by the ultrasounds of a sonicator in a bath for 10 - 20 minutes.

After being dried, the Au and Fe nanoparticles of this preferred aspect of the invention can be stored in a protective atmosphere, at -20 ° C, for a period of more than 12 months, and then be used at any time after the synthesis, just after resuspension in PBS assisted by ultrasound of a sonicator in a bath for 10 - 20 minutes.

The final average composition of the nanoparticles is such that the Au is present in the nanoalloy in an atomic percentage not higher than 80% and not lower than 15%, and the Fe is present in the nanoparticles in a percentage not lower than 20 atomic% and not higher than 85%. The achievement of alloy nanoparticles within these composition ranges was demonstrated by elemental analysis with "inductively coupled plasma assisted mass spectroscopy" and elemental analysis with the scanning electron microscope and probe for energy dispersion spectroscopy (SEM-EDX), as demonstrated in the experimental part (Example 1, Fig. 2).

In another preferred embodiment of the present invention, the metal nanoparticles with a geometric size between 1 and 1000 nm, preferably between 1 and 500 nm comprise a metal nanoalloy where X is Fe, Y is B and Z is absent, where X is present in an atomic percentage between 20% and 95% and Y is present in an atomic percentage between 5% and 80%, and a biocompatible organic stabilizer.

These nanoparticles are produced through a synthesis method that acts in conditions far from thermodynamic equilibrium, as this Fe-B metal nanoalloy is not thermodynamically stable at ambient pressure and temperature, except for a few specific compositions.

For example, the nanoparticles are obtained by laser ablation in liquid, according to the scheme shown in Fig. 1. The laser used in this specific embodiment emits pulses with a duration of 1 - 50 ns and at a wavelength of 1064 nm, with an energy per pulse between 10 and 150 mj. It should be noted that other pulsed laser sources, with different characteristics in terms of duration, wavelength and energy per pulse, can also be used for the same purpose.

The solvent used in this embodiment is anhydrous acetone, with volumes ranging from 100 to 500 mL for each synthesis. Polyvinylpyrrolidone is dissolved in anhydrous acetone with a concentration between 0.005 and 0.5 mg/ mL.

To obtain these nanoparticles in Fe and B metal nanoalloys, other organic solvents can also be used, preferably low molecular weight alcohols, ethers and / or ketones, even more preferably ethanol, isopropanol, tetrahydrofuran and / or acetone, both in ambient atmosphere, and in a controlled atmosphere of inert gas to limit the oxidation of the transition metals present in the nanoalloy.

Preferably, the laser ablation process for obtaining the Fe and B metal alloy nanoparticles is performed in a controlled atmosphere of inert gas.

Polyvinylpyrrolidone has a molecular mass between 10000 and 300.000 Da. It should be noted that other biocompatible organic stabilizers can also be used, such as for example but not limited to polyethylene glycol, cellulose, polyvinyl alcohol, pullulan and dextran.

The introduction of the biocompatible organic stabilizer is essential to provide colloidal stability, and it is necessary to obtain complete dispersion in the aqueous solution after the removal of the organic solvent.

The metal target is made of an alloy composed of Fe and B in a proportion between 80:20 and 10:90 in molar / atomic ratio, and obtained by conventional metallurgical techniques.

The Fe and B alloy nanoparticles of this preferred aspect of the invention are preferably obtained by laser ablation in liquid. They are collected and separated from the liquid by centrifugation at speeds between 100 and 30,000 ref for times between 5 and 120 minutes, or by placing a permanent magnet under the container for 6 - 24 h, to attract all the nanoparticles in the proximity of the magnet and be able to remove the supernatant. The process is then followed by drying at 20 - 30 ° C or lyophilization of the sediment, storage at -20 °C in a protective atmosphere, or resuspended in the desired aqueous solution, such as for example phosphate-buffered saline (PBS), by ultrasonication with a bath or tip sonicator for 10 to 20 minutes.

After drying, the nanoparticles can be stored in a protective atmosphere, at -20 ° C, and then be used at any time after the synthesis, after resuspension in PBS assisted by ultrasound of a bath sonicator for 10 - 20 minutes.

The final average composition of the nanoparticles requires that Fe is present in an atomic percentage of not more than 95% and not less than 20%, therefore B is present in the nanoparticles in a percentage of not less than 5 atomic% and not more than 80%. The achievement of alloy nanoparticles falling within these composition ranges has been demonstrated by elemental analysis with "inductively coupled plasma assisted mass spectroscopy" and elemental analysis with the scanning electron microscope and energy dispersion spectroscopy (SBM-PDX), as demonstrated in the Bxample 6 (Fig. 16).

In another preferred embodiment of the present invention, the metal nanoparticles with a geometric size between 1 and 1000 nm, preferably between 1 and 500 nm comprise a metal nanoalloy where X is Au, Y is Fe and Z is B, where X is present in atomic percentage between 10% and 55%, Y is present in atomic percentage between 20% and 85% and Z is present in atomic percentage between 5% and 50%, and a biocompatible organic stabilizer.

These nanoparticles are produced by a synthesis method that acts in conditions far from the thermodynamic equilibrium, because the metallic nanoalloy of Au, Fe and B is not thermodynamically stable at ambient pressure and temperature, except in conditions of high dilution of one element in the other, or in the case of a few specific compositions.

For example, the nanoparticles are obtained by laser ablation in liquid, according to the scheme shown in Fig. 1. The laser used emits pulses with a duration of 1 - 50 ns and at a wavelength of 1064 nm, with an energy per pulse between 10 and 150 mj. It should be noted that other pulsed laser sources, with different characteristics in terms of duration, wavelength and energy per pulse, can also be used for the same purpose.

The solvent used can be ethanol, with volumes ranging from 100 to 500 mL for each synthesis. As a biocompatible organic stabilizer, thiolated polyethylene glycol (PEG-SH) is dissolved in ethanol with a concentration between 0.005 and 0.5 mg/ mL.

To obtain these nanoparticles in Au, Fe and B metal alloy, other organic solvents can also be used, preferably low molecular weight alcohols, ethers and / or ketones, even more preferably ethanol, isopropanol, tetrahydrofuran and / or acetone, both in the atmospheric environment, and in a controlled atmosphere of inert gas, to limit the oxidation of the transition metals present in the nanoalloy.

According to a preferred aspect, the laser ablation process to obtain the Au, Fe and B metal nanoparticles is carried out in an inert gas atmosphere.

The thiolated polyethylene glycol has a molecular mass between 100 and 30,000 Da. Other biocompatible organic stabilizers can also be used to coat the Au, Fe and B alloy nanoparticles of this preferred aspect of the invention, such as for example but not limited to polyvinylpyrrolidone, cellulose, polyvinyl alcohol, and biocompatible thiolated molecules such as for example but not limited to glutathione, mercaptopropionic acid, mercaptopropansulfonate or mercapto phenyl boronic acid.

The biocompatible organic stabilizer is useful to give colloidal stability in biological fluids and to obtain the complete dispersion of the nanoparticles in the aqueous solution after the removal of the organic solvent.

The metal target used in the laser ablation in liquid synthesis is made of an alloy composed of Au, Fe and B in a proportion between 15:70:15 and 25:60:15 in a molar / atomic ratio and obtained through conventional metallurgical techniques.

The Au, Fe and B nanoparticles of this preferred aspect of the invention are preferably obtained by laser ablation in liquid, then the resulting solution is concentrated by a rotary evaporator, and the nanoparticles are collected and separated from the liquid by centrifugation at speeds ranging from 100 and 30,000 ref for times between 5 and 120 minutes. The process is then followed by drying of the sediment at 20 °C or lyophilization, and conservation in a protective atmosphere at -20 °C or resuspension in the desired aqueous solution, such as the phosphate-buffered saline (PBS), assisted by the ultrasounds of a bath sonicator for 10 - 20 minutes.

After drying, the nanoparticles can be stored in a protective atmosphere, at -20 °C, to then be used at any time after synthesis, after resuspension in PBS assisted by ultrasound of a bath sonicator for 10 - 20 minutes. The final average composition of the nanoparticles requires that the Au is present in the nanoalloy in atomic percentage not higher than 55% and not lower than 10%, the Fe is present in a measure not higher than 85% and not lower than 20%, B is present in the nanoparticles in a percentage of not less than 5 atomic% and not more than 50%. The achievement of alloy nanoparticles falling within these composition ranges has been demonstrated by elemental analysis with "inductively coupled plasma assisted mass spectroscopy" and elemental analysis with the scanning electron microscope and energy dispersion spectroscopy (SEM-EDX) as demonstrated in the Example 10 (Fig.27).

Advantageously, the metal alloy nanoparticles of the present invention can provide quantitative information on the accumulation of sensitizer in the areas to be treated, through MRI or CT scan. This translates into the possibility of calibrating the dose of radiation to be used for therapy, point by point in the area to be treated.

Where this multifunctionality also includes the possibility of increasing the contrast for two different visualization techniques (MRI and CT), the additional advantage is that of combining selective viewing modes for low-density tissues (MRI) with those more selective for tissues at high density (CT), in precisely the same area, and without duplicating the administration of contrast agents for the different techniques.

Where this multifunctionality also includes sensitization for two different radiotherapy techniques (XRT and NCT), the additional advantage is to perform combined treatments to maximize efficacy, reducing the overall dose of ionizing radiation (X-rays and neutrons) compared to a single-type treatment, but achieving the same efficacy with fewer side effects.

The metal nanoparticles of the present invention are characterized by the high efficacy of their multiple functions, due to the high concentration of functional elements for diagnostic or therapeutic purposes.

The good colloidal stability of the metal nanoparticles of the present invention allows effective distribution in the tissues to be treated and limited accumulation in systems or organs responsible for removing foreign bodies from the living organism. Where the chemically degradable nature of the nanoparticle exists, the interaction with the organs responsible for the removal of foreign bodies from the living organism is diluted over time scales ranging from days to weeks, rather than being concentrated in the first hours following administration.

At the same time, it is avoided the immediate removal of the substance by the kidney or its accumulation in the liver already a few hours after administration, allowing instead the preferential accumulation in the tissues to be treated, without resorting to the administration of massive quantities of substance for achieving a comparable accumulation, and thus avoiding overload of the kidneys and liver. This also results in significantly shorter infusion and administration times.

The metal nanoparticles of the present invention comprise only biocompatible substances or substances with tolerable toxicity at the doses used, such as Au, Ag, Fe, B, Mn. In particular, they do not contain gadolinium, which eliminates the risk of nephrogenic systemic fibrosis (NSF) in patients with acute or chronic severe renal insufficiency or acute renal insufficiency of any severity due to hepato-renal syndrome or in the perioperative hepatic transplant period.

Therefore, an object of the present invention is the combined, simultaneous and / or spatially coincident use of the metal nanoparticles described above as contrast agents in diagnostic techniques based on the acquisition of images of the human and / or animal body and as sensitizing agents in radiotherapy techniques.

Preferably, the diagnostic techniques based on the acquisition of images of the human and / or animal body in which the metal nanoparticles of the invention can be used are MRI and / or CT.

Preferably, the radiotherapy techniques in which the metal alloy nanoparticles of the invention can be used are X-ray radiotherapy (XRT) and / or radiotherapy by neutron capture (NCT).

In the following, detailed examples of the metal alloy nanoparticles of the present invention and of their synthesis will be provided, with particular reference to the attached figures 1-35.

The invention thus conceived is susceptible of numerous modifications and variations, all falling within the scope of the present invention as it results from the attached claims.

EXAMPLES

Example 1

Synthesis of nanoparticles in alloy of Au-Fe 1:1.

130 mL of HPLC-grade ethanol >99.8% are measured with a graduated cylinder. The required amount (0.0113 g) of PEG-SH 5000 Da is weighed and added to the EtOH previously prepared in the cylinder. The solution is subjected to ultrasound in a bath sonicator until the PEG is completely dissolved. A metal target 1 cm wide and with composition Au:Fe 25:75 in atomic ratio is inserted in a glass ablation cell.

As indicated above, the depletion in Fe of the final Au-Fe alloy nanoparticles compared to the composition of the bimetallic target used for synthesis, is due to the oxidation of iron and the formation of non-metallic compounds. These substances are separated from the alloy nanoparticles by the purification and washing procedure as described in the text.

The solution of EtOH and PEG-SH is added to the cell. Then a magnetic stir bar is inserted and the solution is shaken vigorously for a few minutes to make it as homogeneous as possible. Finally, a slow magnetic stirring is set.

The parameters of a Nd: YAG Quantel Brilliant laser are set as follows: delay between q- switch and flashlamp = 260 pS, frequency 50 Hz, wavelength 1064 nm, duration of the laser pulse 6 ns, pulse energy laser 60 mj, distance between target and focusing lens (f = 100 mm) equal to 8.5 cm. The target scan under the laser beam is set using a Standa translator controlled by a Labview program. The synthesis is then performed for the necessary time (typically 5 hours). After 5 hours of synthesis, the solution is transferred into a container and stored at -20 °C for 24 h. After this time, the solution is transferred into 50 mL plastic centrifuge tubes and centrifuged for 15 - 30 minutes at 1000 - 1500 ref and a temperature of 5 °C. The supernatant is then removed, and the sediment is washed by centrifugation with a 4/1 methanol/ ethanol mixture for 15-30 minutes at 1000 - 1500 ref and a temperature of 5 °C. The sample is then dried at 20 - 30 °C and stored in a protective atmosphere at -20 °C until its use.

Example 2

Structural characterization of the Au-Fe nanoparticles of Example 1.

The Au-Fe nanoparticles of Example 1 were studied by elemental analysis with "inductively coupled plasma assisted mass spectroscopy" (resulting in Au:Fe 1:1) and elemental analysis with the scanning electron microscope and energy dispersion spectroscopy (SEM-EDX). Figure 2 shows the results of the semiquantitative EDX analysis performed on Au-Fe nanoparticle powder, demonstrating the presence of Au and Fe, in addition to C and O, the latter being typical of the coating with PEG-SH.

The introduction of the biocompatible organic stabilizer is essential to provide colloidal stability to the nanoparticles, and it is necessary to obtain complete dispersion in the aqueous solution after the removal of the organic solvent. As shown in Fig. 3, the resulting colloid is stable in bovine serum (FCS), even after 24 hours, with an average hydrodynamic dimension of 10 nm, measured by DLS technique.

Fig. 4 shows the result of Fourier transform infrared spectroscopy (FTIR) performed on a nanoparticle pellet of Example 1, whose resulting peaks in the range 800 - 1500 cm ¹ and 2800 cm ¹ confirm the coating of the nanoparticles with the polymer.

Fig. 5 shows an image taken by a transmission electron microscope (TEM) of the nanoparticles. Their shape is substantially spherical and with geometric dimensions in a range from 1 to 60 nm, as shown in Fig. 6.

In Fig. 6 it is also shown how this size distribution undergoes a measurable evolution when the nanoparticles are dispersed in a biological fluid such as FCS at a lysosomal pH (equal to 4.5), such that the average size (<10 nm) it is reduced below the optimal threshold for effective removal of foreign bodies from living organisms. This is due to the progressive dissolution of the nanoparticles, as shown in the TEM images of the same sample analyzed after 2 weeks and 2 months of permanence in the FCS solution at pH 4.5 (Fig. 5).

X-ray diffraction (XRD) performed on a powder sample of nanoparticles of Example 1 (Fig. 7) exhibits the characteristic reflections of the F.C.C. of Au, but at different angles corresponding to the formation of an Au-Fe alloy where the Fe atoms occupy the sites of the crystalline lattice according to a random distribution, as inferable from the relative position and intensity of the peaks in the diffraction spectrum. The width of the peaks also indicates a highly defective and disordered crystal structure, attributable to the metastable nature of the Au-Fe alloy.

Example 3

Functional characterization of the Au-Fe nanoparticles of Example 1.

The Au-Fe nanoparticles of Example 1 were analyzed to evaluate their use as a bimodal contrast agent. The contrast for MRI was measured using test tubes containing solutions of nanoparticles with increasing concentration, as shown in Fig. 8, where it is evident the contrast effect on the images weighted according to the transverse relaxation time T2. From the graph of the transverse relaxivity values ¾ = T2-¹ as a function of the Fe concentration in the samples (Fig. 9), and considering the linearity range at lower concentrations, the corresponding transverse relaxivity value is measured, which is equal to 22 mM(Fe)^-1 s^_1. The contrast for TAC is measured in tubes containing solutions of nanoparticles with increasing concentration, as shown in Fig. 10. The X-ray attenuation capacity is measured by the graph of Hounsfield units (HU) as a function of the concentration of Au in the tubes, as shown in Fig. 11, and agrees with what is expected for Au-based compounds. The CT contrast directly measures the ability of the medium to absorb X-rays, therefore the result of Fig. 11 indicates that the sample increases the absorption of X-rays. It is known in the state of the art that gold nanostructures generate Auger electrons following the absorption of X-rays, which have a linear energy transfer coefficient (LET) higher than the X-rays themselves. This makes the Au-Fe nanoparticles of Example 1 sensitizers for XRT.

The invention can therefore be used to increase the contrast of images acquired with any MRI and CT tomograph available on the market for clinical or pre-clinical research use, and to perform XRT of the displayed area.

Example 4

Demonstration of the possibility of using the Au-Fe nanoparticles of Example 1 as contrast agents in vivo.

Figures 12 and 13 report the results of the CT analysis performed using a scanner for small animals with the following acquisition parameters: tube voltage 80 kVp, current 3 mA, 300 sections, voxel dimensions 0.1 mm. The images were reconstructed using the Feldkamp algorithm for CT cone beam. The images are obtained from healthy C57 black 6 mice maintained at 37 °C and under gas anaesthesia (2-3% isoflurane and 1 1/ min of oxygen), after injection of 0.200 mL of Au-Fe nanoparticle solution into PBS in the caudal vein, at a concentration equal to 213 mg_Au/kg_mouse. Image analysis (using imagej) was performed by placing 5 different regions of interest (ROI) on the corresponding organ and the mean Hounsfield unit (HU) values of each ROI were calculated. Figures 12 and 13 show, respectively, the representative images of the spleen and liver of one of the treated animals, at three different time-points, and the mean HU values at the different time points. The increase in contrast, which can be measured as a greater HU value in the regions of interest, clearly indicates the ability of the Au-Fe nanoparticles to act as contrast agents for CT. Furthermore, it is observed how the contrast increases over time, over a few days, thus demonstrating the ability of the Au-Fe nanoparticles to gradually interact with the organs responsible for the clearance of external substances, without immediate accumulation already in the first hours after injection. Overall, this feature is also found in the fact that the increase in contrast in the spleen is higher than in the liver. Fig. 14 shows the MRI analysis performed in vivo on healthy C57 black 6 mice with a tomograph operating at 7 T. The animals were injected intravenously with Au-Fe nanoparticles at a dose of 5 mg_Fe/kg_mouse. For MRI imaging, the animals were anaesthetized with gas anaesthesia (a mixture of O2 and air containing 1-1.5% isofluorane), placed in a heated bed and placed in a coil with an internal diameter of 7.2 cm. T2-weighted body images of mice were acquired using a rapid relaxation acquisition (RARE) sequence with the following parameters: FOV = 60 x 40 mm, MTX = 256x256, slice thickness = 1 mm, TE = 33 ms and TR = 2,500 ms. As can be seen in Fig. 5, the presence of a clearly measurable contrast indicates the ability of the Au-Fe nanoparticles to act as MRI contrast agents and, therefore, to be bimodal contrast agents given also the results of CT in vivo. Furthermore, the image contrast and variation of the mean T2- weighted signal in the regions of interest (liver and spleen) agree with the CT analysis and indicate the slow interaction with these organs, aimed at avoiding the overloading in the first days after administration. In particular, unlike the CT analysis, the MRI analysis of the liver does not show a peak of accumulation after the first week, indicating that the particles that are blocked in the liver on this time scale are mainly the product of the degradation Au-Fe alloy, resulting predominantly in Au-based nanoparticles.

The ability of Au-Fe alloy nanoparticles to accumulate in tumors was assessed with mice models. Mice were inoculated subcutaneously with 500.000 MB49 tumour cells in the chest wall region. After 7 days, Au-Fe nanoparticles were administered to mice by intravenous injection (0.2 mF at 160 mg Au/kg body weight). The CT images of the tumors were collected at 1 h, 1, 2 and 7 days after injection using an X-ray tube operating at 80 kV. The experiment was repeated also using a reference of pure Au nanoparticles at the same dosage. The measured Hounsfield units (HU) were compared with those of mice not treated with nanoparticles. The uptake of Au-Fe nanoparticles measured in the tumour region is responsible for a significantly higher HU value compared to the control mouse (Figure 15), as well as to the HU before nanoparticles administration (time 0) and to the HU measured in mice treated with the reference Au nanoparticles. The higher HU value in mice treated with Au-Fe NPs persisted for 7 days after nanoparticles administration. This is a desirable feature for the use of Au-Fe alloy nanoparticles as, simultaneously, contrast agents for diagnostic purposes and radiosensitizers for XRT. Concerning the use as radiosensitizer multiple injection of a dose equal to 0.2 mF of 160 mg AuFe/kg body weight can be performed in order to increase the Nanoparticles concentration in the tumour region

Example 5

Synthesis of nanoparticles in alloy of Fe-B 75:25 125 mL of anhydrous acetone (Prolabo, max 0.01% of water) taken by syringe is measured with a graduated cylinder, and the required quantity (0.0125 g) of PVP 40000 Da is weighted and added to the acetone previously prepared in the cylinder. The solution is subjected to ultrasound through a bath sonicator until the PVP is completely dissolved. A metal target 1 cm wide and with an alloy composition of Fe:B 66:34 in atomic ratio is inserted in a glass ablation cell. The acetone and PVP solution is added to the cell. Then a magnetic stir bar is inserted and the solution is shaken vigorously for a few minutes to make it as homogeneous as possible. Finally, a slow magnetic stirring is set. Then a lid with a tube is inserted which serves to insufflate Ar into the solution for all the time necessary for the synthesis.

The parameters of a Nd: YAG Quantel Brilliant laser are set as follows: delay between q- switch and flashlamp = 260 pS, frequency 50 Hz, wavelength 1064 nm, duration of the laser pulse 6 ns, pulse energy laser 60 mj, distance between target and focusing lens (f = 100 mm) equal to 8.5 cm. The scan of the target under the laser beam is set using a Standa translator controlled by a Labview program. Then the synthesis is performed for the necessary time (typically 7 hours). After 7 hours of synthesis, the solution is transferred into a glass bottle and a permanent NdFeB magnet (with dimensions of 3 cm x 3 cm x 1.5 cm) is placed below it, to collect the nanoparticles by magnetophoresis. After 24 h, the supernatant is removed as much as possible with the help of a syringe. The bottom body is then sonicated, transferred to a glass vial. The sample is then dried at 20 - 30 °C and stored in a protective atmosphere at -20 °C until its use.

Bxample 6

Structural characterization of the Fe-B nanoparticles of Bxample 5.

The Fe-B nanoparticles of Bxample 5 were studied by elemental analysis with "inductively coupled plasma assisted mass spectroscopy" (resulting in FeB 75:25) and with the scanning electron microscope and energy dispersion spectroscopy (SBM-BDX). In fact, Figure 16shows the results of the EDX analysis performed on Fe-B nanoparticle powder, demonstrating the presence of Fe and B, in addition to C and O, the latter being typical of the PVP coating.

The introduction of the biocompatible organic stabilizer is essential to provide colloidal stability to the nanoparticles, and it is necessary to obtain complete dispersion in the aqueous solution after the removal of the organic solvent.

As shown in Fig. 17, the resulting colloid is stable in FCS, even after 1 hour from preparation, with an average hydrodynamic dimension of 203 +/- 124 nm, which is equivalent to the dimensions measured in PBS (298 +/- 110 nm), but lower to that of the same preparation dispersed in distilled water (1.9 +/- 1.3 pm). This is an evidence of the fact that the colloidal dispersion of Fe-B nanoparticles is more stable at physiological pH and in biological fluids than at neutral pH in pure water.

Fig. 18 shows the FTIR spectrum collected on a nanoparticle pellet, which exhibits the characteristic absorption peaks of the polyvinylpyrrolidone covering the nanoparticles (region 1100 - 1700 cm ¹ and 800 - 600 cm⁴), confirming the coating of the nanoparticles with the polymer.

Fig. 19 shows a TEM image of the nanoparticles, whose shape is substantially spherical and with geometric dimensions ranging from 1 to 90 nm, as shown in Fig. 20.

In Figures 19 and 20 it is also shown how this dimensional distribution undergoes a measurable evolution when the nanoparticles are dispersed in a biological fluid such as FCS at pH 4.5 (lysosomal pH), such that the average size (<20 nm) is reduced below the optimal threshold for effective removal from the organism.

The X-ray diffraction performed on the nanoparticle powder (Fig. 21) indicates a metallic structure with various characteristic peaks of the Fe-B alloy, superimposed on an intense background attributable to the presence of amorphous phases.

Example 7

Functional characterization of the Fe-B nanoparticles of Example 5.

The action of nanoparticles as a contrast agent for MRI is measured using test tubes containing solutions of nanoparticles with increasing concentration, as shown in Fig. 22, where it is evident the contrast effect on the images weighted on the transverse relaxation time T2. The corresponding transverse relaxivity value ¾ is evaluated, which is equal to 82 mM(Fe)-¹ sX

It is known in the state of the art that boron compounds generate alpha (a) particles and ³Fi⁺ nuclei following the absorption of thermal neutrons, and such a and ³Fi⁺ particles have a significantly higher linear energy transfer coefficient (FET) to the neutrons themselves. This makes the Fe-B nanoparticles sensitizers for NCT. This property has been verified by subjecting increasing quantities of Fe-B nanoparticles to irradiation with thermal neutrons, using neutron activation autoradiography. The number of a particles generated from the sample was compared with those generated under the same conditions by a reference boron compound (H₃BO₃). The result showed that Fe-B nanoparticles generated a number of particles comparable to or greater than those of the standard (Fig. 24). The positive deviation is due to the better dispersion of the nanomaterial on the measuring plate compared to the boron salt. This result indicates that Fe-B nanoparticles have the properties required to be used as sensitizers for NCT.

The invention can therefore be used to increase the contrast of images acquired with any MRI tomograph available on the market for clinical use or pre-clinical research, and to perform NCT on the displayed area.

Example 8

Demonstration of the possibility of using the Fe-B nanoparticles of Example 5 as contrast agents in vivo.

Figures 25 and 26 show the MRI analysis performed in vivo on healthy C57 black 6 mice with a tomograph operating at 7 T. The animals were injected intravenously with Fe-B nanoparticles at a dose of 5 mg_Fe/kg_mouse. For MRI imaging, the animals were anaesthetized with gas anaesthesia (a mixture of O2 and air containing 1-1.5% isofluorane), placed in a heated bed and placed in a coil with an internal diameter of 7.2 cm. T2-weighted body images of mice were acquired using a rapid relaxation acquisition (RARE) sequence with the following parameters: FOV = 60 x 40 mm, MTX = 256x256, slice thickness = 1 mm, TE = 33 ms and TR = 2,500 ms. As can be seen in Figures25, the presence of a clearly measurable contrast indicates the ability of Fe-B nanoparticles to act as contrast agents for MRI and, therefore, to be nanomedicines with a diagnostic function coupled with NCT. Furthermore, the results in terms of image contrast and corresponding mean T2 value in the regions of interest (liver and spleen) indicate a slow accumulation of the nanoparticles in these organs.

Example 9

Synthesis of nanoparticles in alloy of Au-Fe-B 52:22:26.

130 mL of ethanol from HPLC >99.8% are measured with a graduated cylinder and the required amount (0.0113 g) of PEG-SH 5000 Da is weighted and added to the EtOH previously prepared in the cylinder. The solution is subjected to ultrasound in a bath sonicator until the PEG is completely dissolved. A metal target with a side 1 cm and with composition Au: Fe:B 33.3:33.3:33.3 in atomic ratio is inserted in a glass ablation cell.

As already indicated above, the depletion in Fe and B of the final Au-Fe-B alloy nanoparticles compared to the composition of the metal target used for the synthesis, is due to the oxidation of iron and boron and the formation of non-metallic compounds. These substances are effectively separated from the alloy nanoparticles by the purification and washing procedure described in the text.

The parameters of a Nd:YAG Quantel Brilliant laser are set as follows: delay between q- switch and flashlamp = 260 pS, frequency 50 Hz, wavelength 1064 nm, duration of the laser pulse 6 ns, pulse energy laser 60 mj, distance between target and focusing lens (f = 100 mm) equal to 8.5 cm. A lid is placed on the cell and argon is blown through a tube inside the solvent, for the duration of the synthesis. The scan of the target under the laser beam is set using a Standa translator controlled by a Labview program. Then the synthesis is performed for the necessary time (typically 3 hours). After 3 hours of synthesis, the obtained colloidal solution is transferred into a flask with the help of a syringe and is concentrated with the aid of a rotary evaporator, heating to 30 °C. The solution is transferred into a container and stored at -20 °C for 24 h. After this time, the solution is transferred into 50 mL plastic centrifuge tubes and centrifuged for 15 - 30 minutes at 1000 - 1500 ref and a temperature of 5 °C. The supernatant is then removed, and the bottom body is washed by centrifugation with a 4/1 methanol/ ethanol mixture for 15-30 minutes at 1000 - 1500 ref and a temperature of 5 °C. The sample is then dried at 20 - 30 °C and stored in a protective atmosphere at -20 °C until its use.

Example 10

Structural characterization of the Au-Fe-B nanoparticles of Example 9.

The Au-Fe-B nanoparticles of Example 9 were studied by elemental analysis with "inductively coupled plasma assisted mass spectroscopy" (resulting in Au:Fe:B 52:22:26) and elementary analysis with a scanning electron microscope and energy dispersion spectroscopy (SEM-EDX). Figure 26 shows the results of the semiquantitative EDX analysis performed on Au-Fe-B nanoparticle powder, demonstrating the presence of Au, Fe and B, in addition to C and O, the latter being typical of the coating with PEG -SH.

The introduction of the biocompatible polymer is essential to provide colloidal stability to the nanoparticles, and it is necessary to obtain complete dispersion in the aqueous solution after the removal of the organic solvent. As shown in Fig. 28, the resulting colloid is stable in FCS, even after 24 hours, with an average hydrodynamic dimension of less than 10 nm. Fig. 29 shows the FTIR spectrum of a nanoparticle pellet, which exhibits the characteristic absorption peaks of the polyethylene glycol covering the nanoparticles (region 800 - 1500 cm ¹ and 2800 cm ¹). This confirms the coating of the nanoparticles with the polymer.

Fig. 30 shows a TEM image of the nanoparticles. Their shape is substantially spherical and their dimension ranges from 1 to 40 nm, as shown in Fig. 31.

In Fig. 30 and 31 it is also shown how the size distribution undergoes a measurable evolution when the nanoparticles are dispersed in a biological fluid such as FCS at lysosomal pH (pH = 4.5), such that after 2 months the average size (<10 nm) is reduced below the optimal threshold for removing foreign bodies from the living organism.

The X-ray diffraction performed on the nanoparticle powder (Fig. 32) indicates a metallic structure of Au-Fe-B alloy, with crystalline components of F.C.C. cell, as inferable from the relative position and intensity of the peaks that make up the diffraction spectrum. The width of the peaks also indicates a highly defective and partially disordered crystal structure, attributable to the metastable nature of the Au-Fe-B alloy.

Example 11

Functional characterization of the Au-Fe-B nanoparticles of Example 9.

The action of nanoparticles as a contrast agent is twofold. The contrast for MRI is measured using test tubes containing solutions of nanoparticles with increasing concentration, as shown in Fig. 33, where the effect on the images weighted based on the transverse relaxation time T2 is evident. From the plot of the relaxivity G2 as a function of the Fe concentration (Fig. 34), the corresponding transverse relaxivity value ¾ is measured, which is equal to 59 mM(Fe) ¹ s ¹.

The contrast for CT is measured in tubes containing solutions of nanoparticles with increasing concentration, as shown in Fig. 35. The contrast generated by the dispersion of alloy nanoparticles agrees with what is expected for Au-based compounds. The CT contrast directly measures the ability of the medium to absorb X-rays, therefore the result of Fig. 36 indicates that the preparation increases the absorption of X-ray electromagnetic radiation. It is known in the state of the art that gold nanostructures generate Auger electrons following the absorption of X-rays, which have a linear energy transfer coefficient (LET) higher than the X-rays themselves. This makes Au-Fe-B nanoparticles sensitizers for XRT.

It is known in the state of the art that boron compounds generate alpha particles and ³Li⁺ nuclei following the absorption of thermal neutrons, and these a and ³Li⁺ particles have a linear energy transfer coefficient (LET) significantly higher than the neutrons themselves. This makes Au-Fe-B nanoparticles sensitizers for NCT. This property was verified by neutron-activated autoradiography, by subjecting increasing quantities of Au-Fe-B nanoparticles to thermal neutron irradiation, and by comparing the number of a particles generated during the process with a reference consisting of a boron compound (H3BO3). The amount of radiation a emitted by the Au-Fe-B nanoparticles is comparable or higher than that emitted by the boron standard (Fig. ), where the positive deviation is attributable to the better dispersion of the nanomaterial on the measuring plate compared to the boron. This result indicates that Au-Fe-B nanoparticles have the properties required to be used as sensitizers for NCT.

The invention can therefore be used to increase the contrast of images acquired with any MRI and CT tomograph available on the market for clinical or pre-clinical research use, and to perform XRT and NCT on the displayed area.

Claims

1. Nanoparticles with a geometric dimension comprised between 1 and 1000 nm, preferably between 1 and 500 nm, comprising a metallic nanoalloy and a biocompatible organic stabilizer wherein: a) the nanoalloy essentially consists of X-Y-Z, wherein X, Y and Z are different from each other and in which X may be gold , Y may be iron, and Z can be silver, manganese, boron or it may be absent; and b) when Z is present, X varies in an atomic percentage range comprised between 5% and 95%, Y varies in an atomic percentage range comprised between 2.5% and 85% and Z varies in an atomic percentage range comprised between 2.5% and 50%; when Z is absent, X and Y varies in an atomic percentage range comprised between 5% and 95% with the condition that when X is gold and Y is iron, X is present in an atomic percentage comprised between 15% and 80% and Y is present in atomic percentage between 20% and 85%, and characterized in that they are degradable in a biological fluid

2. Nanoparticles according to claim 1, wherein: a) X is gold , Y is iron and Z is boron; and b) X is present in an atomic percentage comprised between 10% and 55%, Y is present in an atomic percentage comprised between 20% and 85% and Z is present in an atomic percentage comprised between 5% and 50%.

3. Nanoparticles according to claim 1, wherein: a) X is gold, Y is iron and Z is absent; and b) X and Y are each present in an atomic percentage equal to 50%.

4. Nanoparticles according to claim 1, wherein: a) X is gold, Y is iron and Z is boron; and b) X is present in an atomic percentage equal to 52%, Y is present in an atomic percentage equal to 22% and Z is present in an atomic percentage equal to 26%.

5. Nanoparticles according to any one of the previous claims, wherein the structure of the metallic nanoalloy is ordered crystalline, disordered crystalline, defective and/or amorphous crystalline, preferably it is a mixed crystalline /amorphous or disordered crystalline structure.

6. Nanoparticles according to any one of the previous claims, characterized in that they are degradable in serum at a pH comprised between 4 and 5.

7. Nanoparticles according to claim 6, characterized by a geometric dimension after degradation comprised between 1 and 60 nm, preferably comprised between 1 and 15 nm.

8. Nanoparticles according to any one of the previous claims, wherein the biocompatible organic stabilizer is a biocompatible polymer having a molecular weight between 100 Da and 300,000 Da, preferably selected from polyethylene glycol, polyethylene glycol thiolate, polyvinylpyrrolidone, polyvinyl alcohol, cellulose, dextran and/ or pullulan.

9. Nanoparticles according to any one of the claims 1 to 8, wherein the biocompatible organic stabilizer is a thiolate molecule, preferably selected from glutathione, mercaptopropionic acid, mercaptopropanesulfonate and/or mercaptophenylboronic acid.

10. Nanoparticles according to any one of the previous claims, obtainable with fast condensation processes of metallic elements in nanophases, at room temperature or in an inert gas atmosphere.

11. Nanoparticles according to claim 10, obtainable through the laser ablation procedure or the laser spray pyrolysis procedure.

12. Nanoparticles according to claim 11, wherein the laser ablation procedure is performed with laser pulses between 800 nm and 1,200 nm.

13. Nanoparticles according to claim 11, wherein the laser ablation process is performed in an organic solvent, preferably in low molecular weight alcohols, ethers and/or ketones, even more preferably in ethanol, isopropanol, tetrahydrofuran and/ or acetone.

14. Nanoparticles according to any one of the previous claims, for the combined, simultaneous and/ or spatially coincident use as contrast agents in diagnostic techniques based on the acquisition of images of the human and/ or animal body and as sensitizing agents in the radiotherapeutic techniques.

15. Nanoparticles for the combined, simultaneous and/or spatially coincident use according to claim 14, wherein the diagnostic techniques based on the acquisition of images of the human and/or animal body are nuclear magnetic resonance and/or computerized axial tomography X-ray.

16. Nanoparticles for combined, simultaneous and/ or spatially coincident use according to claim 14, wherein the radiotherapeutic techniques are X-ray radiotherapy and/or neutron capture radiotherapy.

17. Nanoparticles for combined, simultaneous and/ or spatially coincident use according to claim 14, wherein the use as sensitizing agent for radiotherapeutic techniques is obtained by multiple injection of the nanoparticles