Ratiometric contrast agent for magnetic resonance imaging of temperature

Abstract

The present disclosure relates to a ferrofluid contrast agent composition for measurement of absolute temperature by magnetic resonance imaging, MRI, comprising a magnetic nanoparticle comprising at least one metal atom, or at least one lanthanide atom; and at least a capping agent, wherein the magnetic nanoparticle has a temperature-dependent differential between transverse and longitudinal relaxivity for measuring absolute temperature; or has a temperature-dependent differential between MRI proton resonance frequency and a full-width-half-maximum, FWHM, MRI resonance peak for measuring absolute temperature. The use of said contrast agent as a contrast agent for measurement of absolute temperature by magnetic resonance imaging, as well as its use in medicine, is also disclosed.

Description

D E S C R I P T I O NRATIOMETRIC CONTRAST AGENT FOR MAGNETIC RESONANCE IMAGING OF TEMPERATURETECH NICAL FIELD

[0001] The present disclosure relates to a magnetic contrast agent working as a ratiometric thermometer working in depth and at distance (wireless) for magnetic resonance imaging comprised of nanoparticles with well-defined changes in magnetic susceptibility over a temperature of interest.BACKGROUND

[0002] Nanothermometers are valuable tools for wireless determination of temperature with high space resolution. Currently, most solutions are based in optical properties, which limit their application to surface, particularly in low transparent media, such as tissues. Magnetic nanothermometers based solutions can overcome this limitation, since tissues are transparent to magnetic fields.

[0003] Magnetic Resonance Imaging (MRI) is a technique of choice to readout the temperature information given by the magnetic nanothermometers. However, current MRI nanothermometers give relative temperature variations only, have limited colloidal stability, and still have sensitivity limitations when compared to optical nanothermometers. Therefore, magnetic nanothermometers able to give absolute temperature are needed.

[0004] Magnetic resonance imaging is a well-established diagnosis technique. MRI apparatus use strong magnetic fields, magnetic field gradients and radio waves to generate MRI-images. The contrast between different tissues is determined by the rate at which excited atoms return to the equilibrium state (relaxation). By varying the parameters of the pulse sequence, different contrasts may be generated between tissues based on the relaxation properties of the hydrogen atoms therein. For certain applications, contrast agents may be used to provide better contrast imaging, most of them being based on chelates of gadolinium.

[0005] MRI thermometry is a medical and research technique able to detect changes of temperatures in a body, organ, or tissue, that may occur by induction of external source, such in a thermal treatment, or internally, such as the changes of local temperature produced by a tumour. Different methods and techniques for temperature determination using MRI have been described. These methods can be grouped in two categories: methods free of contrast agents and methods using contrast agents.

[0006] The methods free of contrast agents have sensitivity limitations and all of them provide information about temperature variation and not information about absolute temperature. Among free contrast agent methods for temperature determination, the one based on the water proton frequency shift is the most explored one. No absolute temperature is obtained with this method since the water proton frequency depends critically on hydrogen bonds and thus varies from tissue to tissue. This variation from tissue to tissue implies that the methods free of contrast agents only provide information about temperature variation and not information about absolute temperature. At the same time, they present sensitivity limitations associated to the inherent small temperature dependence of the water resonance frequency (between - 0.006 ppm °C1to -0.01 ppm °C’1).[1]

[0007] The methods based on contrast agents include thermosensitive materials (e.g., liposomes) releasing a temperature-insensitive contrast agent and interchange of water molecules. [2] The methods based on contrast agents may also include temperaturesensitive contrast agents. Temperature-sensitive MRI contrast agents based on ferromagnetic nanoparticles [3,4], doped cobalt ferrite (CoFezC ) nanoparticles [5], and paramagnetic liposomes loaded with lanthanide or gadolinium-based complexes are known in the art. These methods are useful to indicate the cross of a temperature threshold and not to give an absolute temperature measurement.

[0008] As example, the use of solid gadolinium metal, transitioning from a ferromagnetic state to a paramagnetic state around room temperature, promotes a temperature-dependent transverse relaxivity of hydrogen nucleus. This method is not ratiometric and thus depends on the proximity between metal and sample, the geometry and relative quantities (metal concentration in the tissue). [6] In another example, [7] an evolution from gadolinium to doped soft ferrite nanoparticles waspresented, but the system is still not ratiometric, depending on the nanoparticles concentration which is unknown in practical applications. [7] At the same time, the system is composed of aggregates of crystallites with 210 nm, with difficult colloidal stability which is crucial for practical biomedical applications.

[0009] While the determination of a temperature variation, as proposed using the above-mentioned systems, can already give valuable information is several contexts, absolute temperature is the target information to detect abnormal function or to monitor and control hyperthermia procedures such as tumour ablation. Measure and monitor absolute temperature can lead to better clinical outcomes. Such self-referenced thermometers give a superior information about temperature, much less prone to variations, such as variations in concentration, or variations in the relaxivity properties of tissues. This can be useful, for instance, on tumour ablation procedures where it is needed an increase in temperature up to a given value within a well-defined temperature range, where excess for temperature leads to carbonization and a temperature below that range can have a nefarious effect, such as the promotion of metastasis.

[0010] Document EP3631455 describes a probe element for separation and sensing of analytes of interest controlled by temperature. The probe element includes at least one magnetic crystal and one or more types of capping agents. The magnetic crystal produces a stable magnetic field at the temperature of interest for sensing or separation. The stable magnetic field can be controlled by temperature and the probe can be integrated in a sensing and or separation device and process. However, this document does not disclose a magnetic contrast agent working as a ratiometric thermometer able to give absolute temperature measurements, while having a high sensitivity at physiologic temperature range.

[0011] Document EP2447689 describes a ratiometric optical nanothermometer that may include a magnetic nanoparticle used as a local source of heat. However, this document does not disclose a ratiometric nanothermometer based on magnetic properties or based on a contrast agent.

[0012] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0013] The present disclosure relates to a colloidal-stable magnetic thermometer able to give absolute temperature measurements while having a high sensitivity at physiologic and apoptosis / necrosis temperature ranges.

[0014] For the scope and interpretation of the present disclosure, the term "physiologic temperature range" refers to temperatures ranging from 36 to 38°C. The term "apoptosis / necrosis temperature range" refers to temperatures higher than 38 °C, preferably from 39 to 60°C.

[0015] The present disclosure describes a contrast agent for measurement of absolute temperature in biological tissues by MRI, exploring the self-referenced temperature given by the contrast agents. Self-referenced thermometers give a superior information about temperature, much less prone to variations in concentration, or variations in the relaxivity properties of tissues, for instance.

[0016] In an embodiment, the contrast agent can be used as a thermometer during a thermal tumor ablation process, or hyperthermia treatments.

[0017] The present application relates to a contrast agent giving absolute temperature values in MRI, comprising at least one magnetic nanoparticle and at least one capping agent.

[0018] For the scope and interpretation of the present disclosure, the term "capping agent" refers to binding molecules that are used in minute quantity for the synthesis of nanoparticles and ferrofluids. These molecules mainly modulate the surface chemistry, morphology, and size distribution of the nanoparticles, stabilize and shield them to prevent agglomeration in the ferrofluid.

[0019] In an embodiment, the magnetic nanoparticle produces a temperature dependent magnetic susceptibility which in turn affects the transverse and longitudinal relaxivity of neighboring hydrogen nuclear spins in a different way. In particular, the magnetic nanoparticle produces a temperature dependent magnetic susceptibility that affects strongly the transverse relaxivity of neighboring hydrogen nuclear spins and has a small effect on the longitudinal relaxivity.

[0020] For the scope and interpretation of the present disclosure, the term "magnetic susceptibility" is regarded as a dimensionless proportionality constant that indicates the degree of magnetization of a material in response to an applied magnetic field. It indicates whether, and how much, a material is attracted into or repelled out of a magnetic field.

[0021] The ratio between the transverse and longitudinal relaxivity of neighboring hydrogen nuclear spins gives a measurement of absolute temperature, which is independent of the concentration of the magnetic nanoparticle.

[0022] In an embodiment, the magnetic nanoparticle is doped with a radioactive tracer, allowing multimodal magnetic resonance imaging and positron emission tomography.

[0023] In an embodiment, the magnetic nanoparticle includes a at least one paramagnetic capping agent, such that the magnetic nanoparticle produces a temperature dependent magnetic field that affects strongly the transverse relaxivity of neighboring hydrogen nuclear spins. The paramagnetic capping agent has a temperature-independent effect on the longitudinal relaxivity of neighboring hydrogen nuclear spins. Thus, the ratio between the transverse and longitudinal relaxivity of neighboring hydrogen nuclear spins gives a measurement of absolute temperature independent of the concentration of the magnetic nanoparticle.

[0024] In an embodiment the at least one capping agent contributes to the stabilization of the nanoparticles in aqueous media.

[0025] In another embodiment at least one of the capping agents is an anchoring agent, used to target and anchor to specific molecules or a specific location.

[0026] For the scope and interpretation of the present disclosure, the term "anchoring agent" refers to a molecule or macromolecule that actively targets and binds to a specific molecule.

[0027] In an embodiment, the at least one capping agent is an organic compound, inorganic compound, or a mixture thereof.

[0028] The present disclosure relates to a ferrofluid contrast agent composition for measurement of absolute temperature by magnetic resonance imaging, MRI, comprising: a magnetic nanoparticle comprising at least one metal atom, or at least onelanthanide atom; at least a capping agent, wherein the magnetic nanoparticle has a temperature-dependent differential between transverse and longitudinal relaxivity for measuring absolute temperature; or has a temperature-dependent differential between MRI proton resonance frequency and full-width-half-maximum, FWHM, MRI resonance peak for measuring absolute temperature.

[0029] In an embodiment, the temperature-dependent differential between transverse and longitudinal relaxivity is provided by a temperature-dependent magnetic susceptibility of the magnetic nanoparticles.

[0030] In another embodiment, the temperature-dependent differential between MRI proton resonance frequency and full-width-half-maximum, FWHM, MRI resonance peak is provided by a temperature-dependent magnetic susceptibility of the magnetic nanoparticles.

[0031] In an embodiment, the magnetic susceptibility can be obtained my measuring the relaxivity rl, and r2, and observe if they have different variations with temperature. The relaxivities ri and r2 at a given temperature and at a given external magnetic field are determined in a relaxometer with control of temperature and field. Briefly, the ferrofluid with a known concentration of nanoparticles is placed in a solid container under an external magnetic field and at a constant temperature. An inversion recovery sequence is used to determine ri and a Carr-Purcell-Meiboom-Gill (CPMG) sequence is used to determine r2.

[0032] In another embodiment, the magnetic susceptibility can be obtained by measuring the proton resonance frequency (prf) and the full width at half maximum (FWHM) of the resonance peak.

[0033] In an embodiment, one of the measured parameters (either relaxivity rl, or r2; or FWHM or prf, respectively) has a small temperature-dependent variation, and serve as a reference.

[0034] In an embodiment for better results, the magnetic nanoparticle has a temperature-dependent magnetic susceptibility within the temperature range from 5 and 60 °C.

[0035] In an embodiment, the magnetic nanoparticle is a ferrimagnetic nanoparticle.

[0036] In an embodiment for better results, the magnetic nanoparticle is an iron oxide nanoparticle.

[0037] In an embodiment for better results, the magnetic nanoparticle is a doped epsilon iron oxide nanoparticle.

[0038] In an embodiment for better results, the magnetic nanoparticle is a hard magnet. Hard magnets have a steeper temperature dependence of the magnetic susceptibility, unlike superparamagnetic nanoparticles whose temperature dependence is governed by the Curie law having an intrinsic small temperature dependence around room temperature.

[0039] In an embodiment for better results, the magnetic nanoparticle is undergoing a magnetic order-disorder phase transition associated with the temperature-dependent magnetic susceptibility.

[0040] In an embodiment, for better results, the magnetic dipolar interaction energy is lower than the thermal energy, despite the factthat they are hard magnets, contributing to their colloidal stability.

[0041] In an embodiment for better results, the metal atom is Fe, Al, Ga, or mixtures thereof.

[0042] In an embodiment for better results, the lanthanide is gadolinium.

[0043] In an embodiment for better results, the magnetic nanoparticle is a doped epsilon nanoparticle in the form of A^Fez- Ch, wherein x ranges from 0.4 to 0.9 and wherein A is a trivalent cation selected from a list consisting of boron, aluminium, gallium, indium, and combinations thereof.

[0044] In another embodiment, the magnetic nanoparticle is a doped epsilon nanoparticle in the form of A ByFe2-(x+y;O3, wherein A and B are trivalent cations independently selected from a list consisting of boron, aluminium, gallium, indium, and combinations thereof; wherein x ranges from 0.4 to 0.9; y ranges from 0 to 0.9; and wherein A is different from B.

[0045] In an embodiment for better results, the composition is a water-based ferrofluid.

[0046] In an embodiment for better results, the smallest dimension of the magnetic nanoparticles ranges from 5 to 500 nm, measured by transmission electron microscopy.

[0047] In an embodiment, the magnetic nanoparticles are substantially spherical. The substantially spherical magnetic nanoparticles can include spherical or nearly-spherical particles.

[0048] In an embodiment for better results, the smallest dimension of the nanoparticles ranges from 20 to 80 nm, measured by transmission electron microscopy; preferably the smallest dimension of the nanoparticles ranges from 20 to 40 nm, measured by transmission electron microscopy.

[0049] In an embodiment for better results, the concentration of the magnetic nanoparticles ranges from 0.1 mg / mL and 4 mg / mL.

[0050] In an embodiment for better results, the capping agent is a paramagnetic capping agent, preferably a gadolinium chelate; or a polymer; or a silane-based capping agent.

[0051] In an embodiment for better results, the capping agent comprises a reactive group selected from a list comprising carboxylic, amine, hydroxyl, or a thiol group.

[0052] In an embodiment for better results, the magnetic nanoparticle is doped with a radioactive element.

[0053] In an embodiment for better results, the magnetic nanoparticle is an iron oxide nanoparticle doped with radioactive gallium.

[0054] In an embodiment for better results, the magnetic nanoparticle is a doped epsilon GaxFe2- O3 nanoparticle, wherein x ranges from 0.4 to 0.8.

[0055] In an embodiment for better results, the magnetic nanoparticle is a doped epsilon Gao.eFe1.4O3 nanoparticle.

[0056] In an embodiment for better results, the magnetic nanoparticle is an iron oxide nanoparticle doped with aluminium.

[0057] In an embodiment for better results, the magnetic nanoparticle is a doped epsilon AIFezO nanoparticle.

[0058] An aspect of the present disclosure relates to the use of the disclosed contrast agent composition as a contrast agent for measurement of absolute temperature by magnetic resonance imaging or by nuclear magnetic resonance.

[0059] In an embodiment for better results, the contrast agent composition is for use on determination during manipulation of a thermosensitive material.

[0060] The present disclosure also relates to a contrast agent composition for use in medicine.

[0061] In an embodiment, the composition is for use in cancer therapy, preferably hyperthermia or cryogenic therapy.

[0062] In an embodiment, the composition is administrated by oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, or intrathecal route.

[0063] An aspect of the present disclosure relates to a kit comprising the disclosed contrast agent composition, and instructions for temperature determination using magnetic resonance imaging, and / or instructions for administration of the composition to a subject.

[0064] In an embodiment for better results, the contrast agent composition is for use as contrast agent for measurement of absolute temperature by magnetic resonance imaging during or after a hyperthermia or cryogenic treatment.BRI EF DESCRI PTION OF THE DRAWI NGS

[0065] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0066] Figure 1: Schematic representation of A) an embodiment of a magnetic nanoparticle (101) and a capping agent (120); B) an embodiment of a magnetic nanoparticle (101) and a paramagnetic capping agent (121); and C) an embodiment of a magnetic nanoparticle (101), the capping agent (120) and doping atoms suitable for positron emission tomography (130). The arrow inside of the particle (101) represents the magnetic moment of the nanoparticle and the small arrows inside the paramagnetic capping agents (121) indicates the magnetic moment of the paramagnetic agents.

[0067] Figure 2: A) Embodiment of a transmission electron microscopy image, B) size histogram and C) X-ray diffraction pattern of the nanoparticle (101) for compositions Gao.67Fe1.33O3.

[0068] Figure 3: Embodiment of the temperature dependence of the mass magnetization recorded at the external fields 1 kOe, 2.5 kOe and 5 kOe for compositions Gao.67Fe1.33O3 and 6-Gao.77Fe1.23O3

[0069] Figure 4: Embodiment of temperature dependence of A) r? in sample of e- Gao.67Fe1.33O3 recorded at the external fields 0.25 T, 0.5 T and 5 T and B) ri and r? recorded at 1.5 T in the same sample. Based on this dependence it is possible to determine a relative variation, defined as the relative sensitivity of a nanothermometer based on ri or r?.

[0070] Figure 5: Embodiment of the variation of the inverse of the transverse relaxation time (R2=l / T2) and the inverse of the longitudinal relaxation time (Ri=l / Ti). for different temperatures in the 30 to 38 °C range and selected concentrations from ci = 2.2 mM to (l / 10)ci = 0.22 mM for a sample with dextran-coated 6-Gao.5Fe1.5O3 nanoparticles. Full lines represent a temperature variation for selected (constant) concentrations. For each concentration, a large variation of R2(T) with temperature is observed, contrasting to the much smaller variation of Ri(T). Dotted lines represent isothermal lines for selected temperatures.

[0071] Figure 6: Embodiment of temperature dependence of A) the proton resonance frequency and B) of the FWHM of the resonance peak of a sample with dextran-coated 6-Gao.6Fe1.4O3 nanoparticles.

[0072] Figure 7: Embodiment of 2D temperature maps obtained for a ferrofluid containing 6-Gao.eFe1.4O3 nanoparticles inside a plastic Eppendorf vial, at different time points between 0 and 6 min during the cooling of a phantom between the average temperature of 46 °C and the average temperature of 32 °C, based on the variation of R2 and having Ri as a reference, exploring the temperature dependence of T2* ■DETAILED DESCRIPTION

[0073] The present disclosure relates to a ferrofluid contrast agent composition for measurement of absolute temperature by magnetic resonance imaging, MRI, comprising: a magnetic nanoparticle comprising at least one metal atom, or at least one lanthanide atom; at least a capping agent, wherein the magnetic nanoparticle has temperature-dependent differential between transverse and longitudinal relaxivity for measuring absolute temperature; or has a temperature-dependent differential between MRI proton resonance frequency and a full-width-half-maximum, FWHM, MRI resonance peak for measuring absolute temperature. The use of said contrast agent as a contrast agent for measurement of absolute temperature by magnetic resonance imaging, as well as its use in medicine, is also disclosed.

[0074] The present application relates to a contrast agent composition for measurement of absolute temperature by MRI, where the contrast agent composition acts as a sensor, giving information about absolute temperature based on the effect of the contrast agent composition on the transverse and longitudinal relaxivity of neighboring hydrogen nuclear spins and on their different temperature behavior. The determination of absolute temperature can be done at distance and in-depth based on the ratiometric determination of two relaxation times whose distinct temperature dependence is governed by magnetic susceptibility.

[0075] In an embodiment, the composition of the present disclosure comprises magnetic nanoparticles (101) that have several effects on the relaxivity properties of the protons, being at least one of the effects strongly temperature-dependent and at least one of effects weakly temperature-dependent, while no effect is time-dependent. The necessary condition is that the two temperature variations are linearly independent, i.e., they have independent information about temperature.

[0076] In a preferred embodiment, the magnetic nanoparticles (101) have an effect on the transverse relaxivity (T2) that is strongly temperature dependent and have an effect on the longitudinal relaxivity (Ti) that is weakly temperature dependent (Fig. 4 and Fig. 4).

[0077] In an embodiment, the magnetic nanoparticles (101) have an effect on the width of the resonance that is strongly temperature dependent (Fig.6B).

[0078] In another embodiment, the magnetic nanoparticles (101) have an effect on the chemical shift of the resonance that is strongly temperature dependent ( Fig.6A) .

[0079] In an embodiment, the nanoparticles have a size between 5 nm and 500 nm, preferably between 5 and 100 nm, more preferably between 20 and 80 nm, measured by transmission electron microscopy, and have a low toxicity, being thus suitable for biomedical applications.

[0080] In an embodiment, the nanoparticles are from the family of iron oxides. In a preferred embodiment, the nanoparticles are from the family of the doped epsilon iron oxides. In another preferred embodiment, the epsilon iron oxides are doped with gallium, aluminium or combinations thereof.

[0081] In an embodiment, the magnetic nanoparticle (101) is designed such that its magnetic susceptibility has a large variation around a temperature of interest, which in turn induces a large temperature variation of the transverse relaxivity of neighbouring protons, while the longitudinal relaxivity of neighbouring protons has a different temperature dependence, as shown in Fig. 4A. At the same time, both the transverse and longitudinal relaxivity have a similar dependence on the concentration of the magnetic nanoparticle such that the ratio between transverse and longitudinal relaxivities gives an absolute measure of temperature, as shown in Fig. 5.

[0082] In an embodiment, the magnetic nanoparticle has a size, composition and structure chosen such that its magnetic susceptibility changes within the temperature range between 5 and 60 °C. In another embodiment, the magnetic susceptibility changes with temperature but not with time within the time range between 1 microsecond and 1 h.

[0083] In an embodiment, the composition comprises at least one transition metal atom or ion, such as iron. In another embodiment, the composition comprises at least one lanthanide atom or ion. In another example, the composition comprises at least one metal or lanthanide ion and one ion of group 16 or 17 of the periodic table, such as gadolinium.

[0084] In an embodiment, the composition comprises iron and aluminium. In another embodiment, the composition comprises iron and gallium.

[0085] In one embodiment, the structure of the magnetic nanoparticles comprised in the disclosed composition is orthorhombic.

[0086] In another embodiment, the magnetic nanoparticle is a polyoxometalate containing at least one transition metal, or at least one lanthanide ion, or at least one ion of group 16 or 17 of the periodic table, or combinations thereof.

[0087] In an embodiment, the magnetic nanoparticle induces a decrease of the transverse relaxivity of neighbouring protons. In another embodiment, the magnetic nanoparticle induces a decrease of the transverse relaxivity of neighbouring protons between 10% and 30% per °C.

[0088] In an embodiment, the magnetic nanoparticle induces a decrease of the longitudinal relaxivity of neighbouring protons. In another embodiment, the magnetic nanoparticle induces a decrease of the longitudinal relaxivity of neighbouring protons between 1% and 10% per °C. In another embodiment, the magnetic nanoparticle induces a decrease of the ratio between the transverse and longitudinal relaxivity of neighbouring hydrogen nuclear spins between 10% 30% per °C.

[0089] In an embodiment, the magnetic nanoparticle (101) is doped with a positron emission tomography contrast agent (130), such as fluorodeoxyglucose, or compounds containing gallium-67. In a preferred embodiment, the magnetic nanoparticle (101) is an iron oxide doped with gallium-67 (67Ga), allowing multimodal magnetic resonance imaging and positron emission tomography.

[0090] In an embodiment, the capping agent (120) is designed as an interface between the magnetic nanoparticle and a surrounding viscous media, such as blood (viscosity around 2.8 mPa.s at 37 °C), providing protection and stabilization. The capping agent (120) may also have an anchoring function, allowing further functionalization of the magnetic nanoparticle. The capping agent (120) may also have an active response, such as reactivity to local conditions such as pH, viscosity, temperature, and reactivity with specific molecules.

[0091] In an embodiment, the capping agent (121) is paramagnetic, decreasing the longitudinal relaxivity of neighbouring hydrogen nuclear spins with a negligible temperature dependence. In another embodiment, the capping agent (121) is a Gd chelate decreasing the longitudinal relaxivity of neighbouring hydrogen nuclear spins.

[0092] In an embodiment the capping agent is a polymer. Examples of polymers used as capping agents include dextran, polyethylene glycol), and polymers containing polyethylene glycol). In another embodiment, the capping agent is a siliceous material obtained after hydrolysis and condensation of alkoxysilanes such as tetraethoxysilane or other organosilanes. In another embodiment the capping agents are chelates, polymers, silica or a mixture thereof.

[0093] In an embodiment the capping agent contains a reactive group such as carboxylic (-COOH), amine (-NH2), hydroxyl (-OH) or a thiol (-SH) group.

[0094] In an embodiment, the disclosed contrast agent composition provides a selfreferenced ratiometric information of temperature. This information can be read wirelessly and in depth by a conventional nuclear magnetic resonance spectrometer (for a zero-dimensional determination) of by a conventional magnetic resonance imaging scanner for a one-, two- or three-dimensional determination of temperature.

[0095] Figure 7 depicts an embodiment where a 2D determination of temperature is obtained after a phantom is homogeneously heated up to 50 °C; the sequence of 2D temperature maps show the subsequent heterogeneous decrease of temperature over time, taken at an initial time point timel= 0 s, and subsequent time points time2 = 10 s, time3 = 68 s, time4 = 103 s, time5=170 s and time6=375 s.

[0096] In an embodiment, the disclosed nanoparticles can be used for temperature determination during or after a hyperthermia or cryogenic treatment, for instance, for tumour ablation. In another embodiment, the disclosed nanoparticles can be used for temperature determination during or after a heat-assisted release of drugs encapsulated on thermosensitive materials.

[0097] Example

[0098] In an embodiment, water-based ferrofluids with e-GaxFe2- O3 nanoparticles were prepared by the hydrolysis and condensation of Ga(lll) and Fe(lll) nitrates in the desired stoichiometric proportion coated by a silica matrix at 1100 °C, followed by the etching of the silica matrix at pH = 11, the dispersion of the isolated nanoparticles at pH = 2, the conjugation with a dextran coating during 12 h at room temperature and the adjustment of pH to a physiologic range.

[0099] In a further embodiment, water-based ferrofluids with <f-GaxFe2- O3 nanoparticles were tested, wherein x = 0.5, x = 0.6 and x = 0.77 were tuned by adjusting the stoichiometric proportions of the nitrites, and using concentrations of nanoparticles between 0.1 mg / mL and 4 mg / mL. The average size of the tested <f-GaxFe2- O3 nanoparticles ranged between 20 and 40 nm when observed by transmission electron microscopy.

[0100] In an embodiment, the Ga and Fe concentration of the ferrofluid was determined by standard coupled plasma atomic emission spectrometry (ICP-AES).

[0101] In an embodiment, the purity of Ga-doped c-Fe2O3 was analysed by standard X-ray diffraction spectroscopy (XRD). The observed peaks match those expected for the crystalline arrangement of the Ga-doped c-Fe2O3 structure and composition (Fig. 2C).

[0102] In an embodiment, the morphology of the nanoparticles was analysed by standard transmission Electron Microscopy (TEM). Briefly, a drop of a ferrofluid is casted on a carbon-coated cupper grid, the solvent evaporates, and the nanoparticles on the grid are imaged in a TEM using acceleration voltages between 100 and 300 kV. TEM images show the existence of orthorhombic-shaped nanoparticles with average size of 39 ± 19 nm (Fig. 2A and 2B).

[0103] The magnetization of the ferrofluids shows a remarkable temperature dependence around room temperature, associated with the disordering of the spins inside each nanoparticle. The temperature at which the disordering occurs, the Curie temperature Tc, decreases with the increase of the Ga doping x, being around 40 and 27 °C forx = 0.6 and 0.7, respectively (Fig.3). Magnetization measurements on the ferrofluid were performed in a field- and temperature-controlled magnetometer using standardextraction method and detection using a squid sensor. The system is calibrated using a Pt cylinder standard (standard reference material 764a, NIST).

[0104] The sharp decrease of magnetization with the increase of temperature around Tc has a noticeable effect on the transverse relaxivity of the protons r2 and a much smaller effect on the longitudinal relaxivity ri, as seen in Fig. 4. The relativities ri and r2 at a given temperature and at a given external magnetic field are determined in a relaxometer with control of temperature and field. Briefly, the ferrofluid with a known concentration of nanoparticles is placed in a solid container under an external magnetic field and at a constant temperature. An inversion recovery sequence is used to determine ri and a Carr-Purcell-Meiboom-Gill (CPMG) sequence is used to determine r2. The relative variation of r2 can be as high as 10% per each degree of temperature variation (Fig. 4) for an external field of 0.5 T. This relative variation is often defined as the sensitivity of the nanothermometer Sr, calculated using Equation (1).where q is the variable used to sense temperature, in this case, r2. A sensitivity of 12% is the highest reported for reversible nanothermometers working in the physiologic range. This relative variation of r2 is field-dependent, since the temperature dependence of magnetization, M(T), is also field-dependent, being smaller as the field increases. This means that, with the increase of the MRI field, the nanothermometers have less sensitivity, decreasing from 10 °C1at 0.5 T to 5 °C1at 1.5 T.

[0105] In an embodiment, the sharp decrease of magnetization with the increase of temperature around Tc (as described above) has also a noticeable effect on the proton resonance frequency and on the full width at half maximum (FWHM) of the resonance peak, with a maximum absolute variation of 0.3 ppm °C’1at 0.5 T and 37 °C. This is at least 30 times higher than the intrinsic variation found naturally in tissues (-0.006 ppm °C’1to -0.01 ppm °C’1), meaning that the Ga-doped e-Fe2O3 nanoparticles give a 30-fold enhanced variation of the current parameter of choice clinically used for the determination of temperature variations. In other words, the use of the disclosed nanoparticles as proton resonance frequency (prf) contrast agents clearly enhance the dynamic range of the prf temperature dependence across the physiologic andhyperthermia range and clearly superposes the inter-tissue prf heterogeneities, making temperature determination by prf using these nanoparticles tissue-independent. The prf and FWHM at a given temperature and at a given external magnetic field are determined in a relaxometer with control of temperature and field. Briefly, the ferrofluid with a known concentration of nanoparticles is placed in a solid container under an external magnetic field and at a constant temperature, the signal of a free induction decay is detected and a Fourier Transformation performed in order to determine the position and FWHM of the Fourier Transformed signal, with reference to the signal of the solvent (water).

[0106] The fact that all current nanothermometers used in MRI give only temperature variations is intrinsically associated to the fact that the measurable quantities that display a temperature dependence (prf, FWHM, Tl, and T2) are, at the same time, temperature- and concentration dependent. This means that absolute temperature is determined only when the concentration of the nanothermometer is known, which does not occur in practical applications where a contrast agent is injected and its final concentration at the site of interest is unknown. To determine the two independent unknown parameters temperature and concentration, we need two linearly independent observable quantities. The noticeable different temperature variation of two independent parameters here disclosed is thus key for a ratiometric absolute temperature determination. The noticeable different temperature variation of 1 / Ti = Ri, and I / T2 = R2, suggest that this pair can be used to determine temperature and concentration at the same time. We can start by generalizing the well-known Ri and R2 dependence on concentration c, allowing them to be temperature-dependent:where / ? (T) and R (7") are the longitudinal and transverse relaxivity of the media where the contrast agent is. The explicit dependence on concentration can be removed, leading to a temperature-only dependency (3)that can be used to unequivocally extract temperature (T) for an unknown concentration c, since, experimentally, it was found that rz(T) is not proportional to ri(T) (Fig. 4). Equation (3) suggests that l / TzfT = constant, c) and l / Ti(T =constant, c) have a linear dependence with temperature with a temperature-dependent slop given by r2(T) / ri(T), which is experimentally found in dextran-coated e-Gao.5Fe1.4O3 nanoparticles (Fig. 5). This slop decreases with the increase of temperature, as expected for a faster decrease of Rz(T, c) with temperature. Together with equation (2), the experimental 1 / T2(T, c) vs. l / Ti(T, c) dependence (shown in Fig. 5 for selected concentrations and selected temperatures (T=31 °C, 35 °C and 38 °C and concentrations (c) between 2.2 mM and 0.22 mM) suggest that each pair of (T, c) values have a unique combination of 1 / T2(T, c) and l / Ti(T, c), such that, by measuring 1 / T2(T, c) and l / Ti(T, c), temperature and concentration can be univocally determined in a given situation where the contrast agent is injected and both of them are unknown. From this point on, further changes of temperature can be determined by measuring both 1 / T2(T, c) and l / Ti(T, c) or, more practically and faster, by measuring just one of temperature-dependent parameters (T2, pfr or FWHM) as usual, but now referenced to the determined point and not just measured as a temperature variation.

[0107] In an embodiment, the determination of concentration and temperature was performed on an Eppendorf phantom using 1 / T2(T, c) and l / Ti(T, c). Samples were heated to around 50 °C, and then the temperature maps were perfumed as a function of time as the phantom cooled back to room temperature by mapping T2* (T) as a faster alternative to map T2. Interestingly, the maps reveal some heterogeneities with the surface cooling faster than the core, as qualitatively expected.

[0108] In summary, water stable doped e-Fe2O3 nanoparticles with a magnetic phase transition around room temperature are temperature-sensitive contrast agents able to provide an absolute measurement of temperature in practical applications where the concentration of the contrast agent is unknown. At the same time, the contrast agents herein disclosed provide the highest reported temperature variations ofT2 and prf together with the lowest size. Thus, the disclosed contrast agent can have an effective application on MRI thermometry, particularly in photothermal therapies using photothermal agents where both agents can be combined.

[0109] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0110] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

[0111] The following claims further set out particular embodiments of the disclosure.References[1] - Rieke V., Pauly K.B., MR Thermometry, Journal of Magnetic Resonance Imaging 27, 376-390, (2008)[2] - Kneidl B., Peller M., Winter G., Lindner, L.H. Hossann, M., Thermosensitive liposomal drug delivery systems: state of the art review, International journal of nanomedicine 9, 4387-4398 (2014)[3] - Settecase F., Sussman M.S., Roberts T.P.L., A new temperature-sensitive contrast mechanism for MRI: Curie temperature transition-based imaging, Contrast Media and Molecular Imaging 2, 50-54 (2007)[4] - Sharma R., Chen C.J., Newer nanoparticles in hyperthermia treatment and thermometry, Journal of Nanoparticle Research 11, 671-689 (2009)[5] - Hankiewicz J.H., Alghamdi N., Hammelev N.M., Anderson N.R., Camley R.E., Stupic K., Przybylski M., Zukrowski J., Celinski Z.J., Zinc doped copper ferrite particles as temperature sensors for magnetic resonance imaging, AIP Advances 7, 056703 (2017)[6] - Hankiewicz, J.H., Celinski, Z., Stupic, K.F., Anderson, N.R., Camley, R.E., Ferromagnetic particles as magnetic resonance imaging temperature sensors, Nature Communications 7, 12415 (2016)[7] - Hankiewicz J.H., Stoll J.A., Stroud J., Davidson J., Livesey K.L., Tvrdy K., Roshko A., Russek S.E., Stupic K., Bilski P., Camley R.E., Celinski Z.J., Nano-sized ferrite particles for magnetic resonance imaging thermometry, Journal of Magnetism and Magnetic Materials 469, 550-557 (2019)

Claims

C L A I M S1. A ferrofluid contrast agent composition for measurement of absolute temperature by magnetic resonance imaging, MRI, comprising: a magnetic nanoparticle comprising at least one metal atom, or at least one lanthanide atom; at least a capping agent, wherein the magnetic nanoparticle has a temperature-dependent differential between transverse and longitudinal relaxivity for measuring absolute temperature; or has a temperature-dependent differential between MRI proton resonance frequency and full-width-half-maximum, FWHM, MRI resonance peak for measuring absolute temperature.

2. The ferrofluid contrast agent composition according to the previous claim wherein the temperature-dependent differential between transverse and longitudinal relaxivity is provided by a temperature-dependent magnetic susceptibility of the magnetic nanoparticles.

3. The ferrofluid contrast agent composition according to the previous claim 1 wherein the temperature-dependent differential between MRI proton resonance frequency and full-width-half-maximum, FWHM, MRI resonance peak is provided by a temperature-dependent magnetic susceptibility of the magnetic nanoparticles.

4. The ferrofluid contrast agent composition according to any of the previous claims wherein the magnetic nanoparticle has a temperature-dependent magnetic susceptibility within the temperature range from 5 and 60 °C.

5. The ferrofluid contrast agent composition according to any of the previous claims wherein the magnetic nanoparticle is a ferrimagnetic nanoparticle.

6. The ferrofluid contrast agent composition according to any of the previous claims wherein the metal atom is Fe, Al, Ga, or mixtures thereof.

7. The ferrofluid contrast agent composition according to any of the previous claims wherein the magnetic nanoparticle is an iron oxide nanoparticle.

8. The ferrofluid contrast agent composition according to any of the previous claims wherein the magnetic nanoparticle is a doped epsilon iron oxide nanoparticle.

9. The ferrofluid contrast agent composition according to any of the previous claims wherein the lanthanide is gadolinium.

10. The ferrofluid contrast agent composition according to any of the previous claims wherein the magnetic nanoparticle is a doped epsilon nanoparticle in the form of A ByFe2-(x+y;O3, whereinA and B are a trivalent cation independently selected from a list consisting of boron, aluminium, gallium, indium, and combinations thereof; x ranges from 0.4 to 0.9; y ranges from 0 to 0.9; andA is different from B.

11. The ferrofluid contrast agent composition according to any of the previous claims wherein the composition is a water-based ferrofluid.

12. The ferrofluid contrast agent composition according to any of the previous claims wherein the smallest dimension of the magnetic nanoparticles ranges from 5 to 500 nm, measured by transmission electron microscopy.

13. The contrast agent composition according to any of the previous claims wherein the smallest dimension of the nanoparticles ranges from 20 to 80 nm, measured by transmission electron microscopy.

14. The contrast agent composition according to any of the previous claims wherein the smallest dimension of the nanoparticles ranges from 20 to 40 nm, measured by transmission electron microscopy.

15. The ferrofluid contrast agent composition according to any of the previous claims wherein the concentration of the magnetic nanoparticles ranges from 0.1 mg / mL and 4 mg / mL.

16. The ferrofluid contrast agent composition according to any of the previous claims wherein the capping agent is a paramagnetic capping agent, preferably a gadolinium chelate; or a polymer; or a silane-based capping agent.

17. The ferrofluid contrast agent composition according to any of the previous claims wherein the capping agent comprises a reactive group selected from a list comprising carboxylic, amine, hydroxyl, or a thiol group.

18. The ferrofluid contrast agent composition according to any of the previous claims wherein the magnetic nanoparticle is doped with a radioactive element.

19. The ferrofluid contrast agent composition according to any of the previous claims wherein the magnetic nanoparticle is an iron oxide nanoparticle doped with gallium.

20. The ferrofluid contrast agent composition according to the previous claim wherein the magnetic nanoparticle is a doped epsilon GaxFe2- O3 nanoparticle, wherein x ranges from 0.4 to 0.8.

21. The ferrofluid contrast agent composition according to the previous claim wherein the magnetic nanoparticle is a doped epsilon Gao.eFe1.4O3 nanoparticle.

22. The ferrofluid contrast agent composition according to any of the previous claims 1-18 wherein the magnetic nanoparticle is an iron oxide nanoparticle doped with aluminium.

23. The ferrofluid contrast agent composition according to the previous claim wherein the magnetic nanoparticle is a doped epsilon AIFezO nanoparticle.

24. Use of the contrast agent composition of any of the previous claims as a contrast agent for measurement of absolute temperature by magnetic resonance imaging or by nuclear magnetic resonance.

25. Use of a contrast agent composition according to the previous claim for temperature determination during manipulation of a thermosensitive material.

26. Contrast agent composition according to any of the previous claims 1-23 for use in medicine.

27. Contrast agent composition according to any of the previous claims 1-23 for use in cancer therapy, preferably hyperthermia or cryogenic therapy.

28. Contrast agent composition for use according to any of the previous claims 26-27 wherein the compound is administrated by oral, parenteral, intramuscular, intranasal, sublingual, intratracheal or intrathecal route.

29. Kit comprising a contrast agent composition according to any of the previous claims 1-23 and instructions for temperature determination using magnetic resonance imaging, and / or instructions for administration of the composition to a subject.

30. Contrast agent composition according to any of the previous claims 1-23 for use as contrast agent for measurement of absolute temperature by magnetic resonance imaging during or after a hyperthermia or cryogenic treatment.

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