Anisotropic contrast agents for magnetic resonance imaging, methods and uses thereof

Abstract

The present disclosure relates to anisotropic magnetic contrast agents containing iron oxide nanoparticles for low-field magnetic resonance scanners, to be applied in the production of morphology- free images, and / or recording / reading information in soft materials containing protons, using low-field magnetic resonance scanners.

Description

D E S C R I P T I O NANISOTROPIC CONTRAST AGENTS FOR MAGNETIC RESONANCE IMAGING, METHODS AND USES THEREOFTECHNICAL FIELD

[0001] The present disclosure relates to magnetic contrast agents for low-field magnetic resonance scanners, to be applied in the production of morphology-free images, and / or recording / reading information in soft materials containing protons, using low-field magnetic resonance scanners.BACKGROUND

[0002] Magnetic resonance imaging (MRI) is a tomographic technique able to produce 3D images of soft tissues and soft materials in a non-invasive fashion, using a uniform external magnetic field, radiofrequency (RF) and gradient fields. While MRI at higher uniform fields result in images with better contrast, low-filed MRI offers good quality images at lower purchase costs, lower ownership costs, with high portability, and possible use in remote areas with no access to liquid helium supply.

[0003] In both high- and low-field magnetic resonance imaging, contrast agents are often used as a minimum-invasive way of increasing contrast, improving the ability to detect features of interest. The ability of a given contrast agent (molecule, nanoparticle or their combination) to generate contrast is measured by its longitudinal and transverse relaxivity. Contrast agents developed so far have an isotropic ability to generate contrast, i.e., have an isotropic longitudinal and transverse relaxivity. These agents include paramagnetic molecules, paramagnetic macromolecules, paramagnetic nanoparticles and superparamagnetic nanoparticles. The final result is an image with a mixture of the signal of the morphological background and signal of the protons affected by the contrast agent. The advantage is that the position of the contrast agent with respect to the morphology is quite well determined; the disadvantage is that the contrast agent may not be clearly discerned from other morphological features that act as a background noise.

[0004] A technique used to separate the contribution of nanoparticles used as contrast agents from the contribution of morphological features is based on the fact that, for larger nanoparticles or higher concentration of nanoparticles, a sufficient resonance shift is generated, and an off-tissue and off-fat resonance image of the nanoparticle neighborhood is produced [1-4], However, this off-resonance signal changes with the aggregation state of the nanoparticles, and the nanoparticles size and concentration should be high enough to produce a shift that is sufficiently large to offset that of fat. At the same time, other effects contribute to this off-resonance signal, including magnetic susceptibility differences at the interfaces. Moreover, this off-resonance technique does not allow a quantitative determination of the contrast agent.

[0005] The 1-3 documents disclose a technique to image only the field created by the contrast agent based on off-resonance imaging, which requires large particles (from micro [1,2] to millimetre size [3]), whereas the present description presents a technique based on the use of much small particles (nanoparticles) with much lower doses, which allows safer procedures.

[0006] The document 3 discloses a technique to image only the field created by the contrast agent based on off-resonance imaging created by a millimetre-sized bead, which requires to turn-off the magnetic field of the MRI scanner before starting the procedures due to the high forces generated by the field gradients when the bead is inserted in the scanner, whereas the present description presents a technique can be applied to MRI scanners with a permanent magnetic field.

[0007] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GENERAL DESCRIPTION

[0008] The present disclosure relates to contrast agents whose ability to generate contrast is not isotropic, unlike all the isotropic contrast agents reported in the state of the art. The disclosed anisotropic contrast can be externally manipulated by an angleand by a magnetic field, allowing an effective separation between contrast agent and morphology contribution and the determination of the contrast agent concentration.

[0009] The present application discloses a contrast agent for low-field magnetic resonance imaging, giving an orientation-dependent relaxivity, comprising at least one magnetic nanoparticle and at least one capping agent. This orientation dependence gives information about the location of the contrast agent without contribution from the morphology, and information about the evolution of the media around the contrast agent, including and not limited to the adhesion state of cells, tissues, and hydrogels.

[0010] In an embodiment, the orientation-dependent contrast agent is used as a probe to image the gelation and dissolution of a hydrogel.

[0011] In an embodiment, the orientation-dependent contrast agent is used as a probe to image the adhesion of cells while forming a tissue or the deadhesion of cells, in particular, the deadhesion of cells due to the action of a protease.

[0012] In an embodiment, the magnetic nanoparticle has a magnetic moment that is not able to align with the magnetic field of the MRI scanner, contributing to an orientation-dependent relaxivity. In particular, the magnetic moment of the nanoparticle must be able to make a 90° angle and a 180° angle with the external magnetic field of the MRI scanner. The orientation-dependent relaxivity can be measured by determining the dependence of the transverse and longitudinal relaxation times with the angle that the magnetic moment of the nanoparticles make with the magnetic field of the MRI scanner. The longitudinal relaxation time can be determined by an inversion-recovery sequence, while the transverse relaxation time can be determined by a CPMG sequence. Nanoparticles exhibiting orientationdependent relaxivity should have a coercivity that is higher than the magnetic field of the MRI scanner. The coercivity of these magnetic NPs can be determined by measuring the field-dependence of the magnetization using a magnetometer.

[0013] Document US20050261575 discloses a method for imaging labelled cells with positive contrast. Spectrally-selective RF pulses are used to excite and refocus the off-resonance water surrounding the labelled cells so that only the fluid and tissue immediately adjacent to the labelled cells are visible in the image.

[0014] An aspect of the present disclosure relates to a composition comprising an embedding agent and an anisotropic contrast agent for low-field magnetic resonance imaging, wherein said anisotropic contrast agent comprises: an iron oxide nanoparticle; a capping agent, wherein the capping agent is selected from a list comprising dextran, polyethylene glycol), organosilanes, chelates, polymers of silica, and mixtures thereof. Surprisingly, the composition and the anisotropic contrast agent of the present disclosure has a direction-dependent transverse and longitudinal relaxivity.

[0015] In an embodiment for better results, the iron oxide nanoparticle is a doped epsilon iron oxide.

[0016] In an embodiment for better results, the iron oxide nanoparticle is doped with AI3+ or Ga3+.

[0017] In an embodiment for better results, the molar ratio between the doping ion and iron ranges from 0 and 0.6; preferably 0 and 0.4.

[0018] In an embodiment for better results, the iron oxide is an iron oxide with formula e-GaxFe2-xO3, wherein x ranges from 0 to 0.5.

[0019] In an embodiment for better results, the iron oxide nanoparticle is a ferrite.

[0020] In an embodiment for better results, the concentration of the contrast agent ranges from 0.001 to 10 mg / mL.

[0021] In an embodiment for better results, the size of the largest dimension of the iron oxide nanoparticle ranges from 20 to 40nm, measured by transmission electron microscopy.

[0022] In an embodiment for better results, the capping agent comprises a carboxylic group, an amine groups, a hydroxyl group, a thiol group, or mixtures thereof.

[0023] Another aspect of the present disclosure relates to the use of a composition as of the present disclosure as an anisotropic contrast agent for magnetic resonance imaging.

[0024] In an embodiment for better results, the composition may be use as an enhancer of contrast for magnetic resonance imaging.

[0025] In an embodiment for better results, the composition may be use as an imaging probe. Preferably, the contribution from the probes is separated from the contribution from the regions where the probes are absent.

[0026] Another aspect of the present disclosure relates to a hydrogel comprising the contrast agent.

[0027] Another aspect of the present disclosure relates to a method for obtaining an anisotropic contrast agent for low-field magnetic resonance imaging described in present disclosure comprising the following steps: incorporation of the contrast agent into a matrix; recording of a suitable direction using an external magnetic field; measuring of an image in two different directions; separating the isotropic and anisotropic components to obtain an image of the distribution of contrast agents in the matrix.BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

[0029] Figure 1: Schematic representation of an embodiment of A) magnetic nanoparticle (101) and capping agents (120); B) magnetic nanoparticles (101) and an embedding agent (130).

[0030] Figure 2A: Representation of an embodiment of results of transverse and longitudinal relaxivity of a magnetic nanoparticle (101) as a function of the angle between the external field of the magnetic resonance scanner and a direction imprinted (recorded) to the magnetic nanoparticle.

[0031] Figure 2B: Representation of an embodiment of results of the transverse relaxivity of a magnetic nanoparticle (101) as a function of the angle between the external field of the magnetic resonance scanner and a direction imprinted to the magnetic nanoparticle by a positive magnetic field (positive imprint) and by a negative magnetic field (negative imprint) with intensity higher than that of the magnetic resonance field and higher than that of the coercive field of the nanoparticle (101).

[0032] Figure 3A: Embodiment of A) 2D spin-echo images obtained in phantoms at angles of 0° and 180° between the magnetic resonance field and imprinted direction. The upper phantoms (R) contain recorded nanoparticles (101) and capping agent (120), while the lower phantoms (NR) contain non-recorded nanoparticles (101) and capping agent (120), serving as a direction-independent control.

[0033] Figure 3B: Embodiment of subtraction between the 2D spin-echo images obtained in phantoms at angle of 0° and angle 180° after a rotation of the image by 180°. In this way, the non-recorded (NR) direction-independent phantom is reduced to noise level, while the recorded (R) direction-dependent contrast agents highlight their presence and location.

[0034] Figure 3C: Embodiment of 2D gradient-echo images obtained in phantoms at angles of 0° and 180° between the magnetic resonance field and imprinted direction. The phantoms contain recorded nanoparticles (101) and capping agent (120).

[0035] Figure 4: Embodiment of the difference between transverse relaxation taken at angles of 0° and 180° between the magnetic resonance field and imprinted direction as a function of the concentration of the nanoparticles (101).

[0036] Figure 5: Embodiment of the angular dependence of transverse relaxation in an alginate hydrogel cross-linked with Ca2+ions and the erase of that angular dependence after the cross-link is broken by the addition of a chelate agent.

[0037] Figure 6: Embodiment of the angular dependence of transverse relaxation in a cellular media where cells are aggregated forming a tissue, the change in angular dependence when a specific direction is recorded by an external magnetic field, an opposite angular dependence when an opposite magnetic field is applied, and theerase of that angular dependence after the cross-link between the cells is broken by the addition of trypsin.

[0038] Figure 7: Embodiment of the angular dependence of magnetization after a preferential direction is recorded.

[0039] Figure 8: Embodiment of hydrodynamic size distribution measured by dynamic light scattering obtained for uncoated and dextran-coated epsilon Gao.2Fe1.8O3 nanoparticles.

[0040] Figure 9: Embodiment of A) transmission electron microscopy images (TEM), B) size histogram and C) X-ray diffraction pattern of the nanoparticle (101) with compositions Gao.2Fe1.sO3 (without capping agent).

[0041] Figure 10: Embodiment of cellular viability after 24 or 48 hours of incubation with different concentrations of magnetic nanoparticles.

[0042] Figure 11: Embodiment of a cell line (L929 cells) cultured in the absence (without NPs) or in the presence of magnetic nanoparticles.DETAILED DESCRIPTION

[0043] The present disclosure relates to magnetic contrast agents for low-field magnetic resonance scanners, to be applied in the production of morphology-free images, and / or recording / reading information in soft materials containing protons, using low-field magnetic resonance scanners.

[0044] The present disclosure relates to contrast agents for magnetic resonance imaging able to produce a morphology-free imaging and to record / read information in low-field magnetic resonance scanners, due to their unique direction-dependent anisotropic relaxivity. These contrast agents comprise at least one magnetic nanoparticle and at least one type of capping or embedding agent.

[0045] The present disclosure relates to a contrast agent for magnetic resonance imaging, MRI, whose relaxivity depends on the angle between a recorded direction and the direction of the MRI magnetic field, comprising a magnetic nanoparticle.

[0046] The present application relates to a contrast agent for MRI able to keep memory and able to show its memory state as a direction-dependent contrast in an MRI image.

[0047] In an embodiment, the magnetic nanoparticle (101) is designed such that its relaxivity depends on the angle between a direction imprinted to the magnetic moment of the nanoparticle (101) and the direction of the magnetic field, as shown in Fig. 2A. This angular dependence provides valuable information about the system, including the location of the magnetic nanoparticles, their concentration, and information about the evolution of the media where the nanoparticles are embedded, including and not limited to cells, tissues, or hydrogels.

[0048] In an embodiment, the magnetic nanoparticles of the contrast agent have a high coercivity and produce a transverse and longitudinal relaxivity on neighbouring hydrogen nuclear spins that depend on the angle between the magnetization of nanoparticle and the external magnetic field of the magnetic resonance scanner. In a preferred embodiment, the magnetic nanoparticles are ferrites with coercivity higher than the external magnetic field of the scanner. In another preferred embodiment, the magnetic nanoparticles are not in a superparamagnetic state, although they are stable in a colloidal suspension. In another preferred embodiment, the magnetic nanoparticles are undoped or doped epsilon iron oxides.

[0049] In an embodiment, the magnetic nanoparticles are not in a superparamagnetic state, such that their magnetization is stable in time.

[0050] In an embodiment for better results, the epsilon iron oxides are doped with Al3+or Ga3+.

[0051] In an embodiment, the molar ratio between the doping ion and iron ranges from 0 to 0.4.

[0052] In an embodiment, the magnetic nanoparticles have between 10 and 100 nm in their largest dimension, as measured by transmission electron microscopy.

[0053] In an embodiment, the direction-dependent relaxivity is used to produce images whose contrast is direction-dependent. In another embodiment, the directiondependent images are produced using spin-echo or turbo spin-echo sequences. Inanother embodiment the direction-dependent images are produced using gradientecho sequences.

[0054] In another embodiment, the presence of the disclosed contrast agent is unequivocally determined, since they are the only elements contributing to the direction-dependent contrast.

[0055] In another embodiment, the direction-independent morphological features of the image are separated from the direction-dependent contrast produced by the agent, resulting in an image of the location of the contrast agent without the presence of the morphological features.

[0056] In another embodiment, the concentration of the contrast agent can be determined from the determination of the relaxivity (Figure 4). In a preferred embodiment, the relaxivity used to determine the concentration of the contrast agent is the transverse relaxivity measured with the magnetic moment parallelly- and anti- pa rallelly aligned with the external field.

[0057] In an embodiment, the value of the direction-dependent relaxivity is controlled by the intensity and direction of an external magnetic field, higher than the magnetic field of the magnetic resonance scanner and higher than the coercive field of the nanoparticles, applied before the magnetic resonance measurements or imaging (Figure 2B). This external magnetic field is used to imprint a preferential direction on the magnetic moment of the nanoparticles and this preferential direction can be detected in a magnetic resonance image. In another embodiment, this external magnetic field is used to record and erase information, whereas the magnetic resonance scanner is used to read this information, in 1, 2 or 3 space dimensions with space resolution.

[0058] In an embodiment, the recorded information is used to detect changes in position and or direction with time of the media where the contrast agent is placed.

[0059] In an embodiment, the recorded information is used to detect changes in viscosity. In a preferred embodiment, the changes in viscosity are detected in hydrogels or tissues. In a preferred embodiment, the changes in viscosity are the result of a gel-sol transition (Figure 5).

[0060] In an embodiment, the recorded information is used to detect changes in the aggregation state of cells. In another embodiment, the change in the aggregation state of cells is a desegregation induced by trypsin or other molecule with similar behaviour (Figure 6).

[0061] The capping agent (120) is designed as an interface between the magnetic nanoparticle and a surrounding media, 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.

[0062] In an embodiment the capping agent is a polymer. In an embodiment for better results, the capping agent is selected from a list comprising dextran, polyethylene glycol), molecules obtained after hydrolysis and condensation of alkoxysilanes such as tetraethoxysilane or other organosilanes, chelates, polymers silica or a mixtures thereof.

[0063] In an embodiment the capping agent contains a reactive group such as carboxylic (-COOH), Amine (-NH2), hydroxyl (-OH) or a Thiol (-SH) group.

[0064] In an embodiment, the magnetic nanoparticle includes a capping agent, acting as a stabilizing agent in aqueous media.

[0065] In another embodiment at least one of the capping agents behaves as an anchoring agent.

[0066] In another embodiment the capping agents are organic molecules / coatings, inorganic molecules / coatings, organic inorganic coatings or mixtures thereof.

[0067] In an embodiment, the embedding agent (130) is designed to encompass several magnetic nanoparticles (101) that will then be delivered together to the designated location. With the embedding agent (130) it is possible to further control the size of the contrast agent and control its sensitivity to the surrounded media. In an embodiment the embedding agent is a polymer. In another embodiment the embedding agent is or contains polyethylene glycol). In another embodiment, the embedding agent is obtained after hydrolysis and condensation of alkoxysilanes suchas tetraethoxysilane or other organosilanes. In another embodiment, the embedding agent contains a polyoxometalate and magnetic nanoparticles (101) its surface.Example:

[0068] The magnetic nanoparticles here described provide information about the media where they are inserted. This information includes their location without a morphological background when using a magnetic resonance scanner, their concentration, and information about the evolution of that media, including rotation of the media, agglomeration and dissociation of cells, formation and viscosity of tissues, formation, evolution and dissociation of hydrogels. All this information can be read wireless and in depth by a conventional low-field nuclear magnetic resonance spectrometer (for an average zero-dimensional determination) of by a conventional low-field magnetic resonance imaging scanner for a one-, two- or three-dimensional imaging.

[0069] In an embodiment, water-based ferrofluids with e-GaxFe2-xO3 nanoparticles, with x=0.2, 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.

[0070] In an embodiment, water-based ferrofluids with e-GaxFe2-xO3 nanoparticles were tested using concentrations of nanoparticles between 1 ug / mL and 1 mg / mL. The average size of the tested e-GaxFe2-xO3 nanoparticles ranged between 20 and 40 nm when observed by electron microscopy.

[0071] In an embodiment, water-based ferrofluids were dispersed in alginate hydrogels. After dispersion, the magnetization is isotropic. However, after placing the hydrogel in a magnetic field higher or equal to 2 tesla, the magnetization becomes anisotropic with a periodic angular dependence, even under an external magnetic field up to 1.5 tesla (Figure 7). This anisotropic angular dependence results in an anisotropic angular dependence of the transverse and longitudinal relaxivity (Figure 2).

[0072] In an embodiment, this anisotropic dependence of the transverse and longitudinal relaxivity is explored to produce images with a direction-dependent contrast (Figure 3). In an embodiment, two images are taken: one at an angle between magnetization and the external field of 0°, and another at an angle of 180°, such that the regions affected by the presence of the anisotropic contrast agent show a different intensity in the two images, while the regions not affected by the presence of the anisotropic contrast agent show the same intensity. This different behaviour is then used to separate the regions affected by the anisotropic contrast agent by the regions not affected by said agent by the subtraction between the image taken at 0° and a 180° rotation of the imagen taken at 180° (Figure 4), since the subtraction in the nonaffected regions results in a background noise, while the subtraction in the contrast agent-affected regions provides an image of these regions.

[0073] In an embodiment, a preferential direction in an alginate hydrogel using an external magnetic field of 2 tesla was recorded, and the same direction is recorded in a 0.5 tesla MRI scanner. Then, changes in the aggregation state and orientation of that hydrogel can be detected, such as a disaggregation induced by the addition of EDTA, that promotes the chelation of the Ca2+ions present in the hydrogel by EDTA, leading to an erase of the recorded direction (Figure 5).

[0074] In an embodiment, e-GaxFe2-xO3 nanoparticles, with x=0.2 and concentration of lpg / mL were added and incorporated into L929 cells (cell line of mouse fibroblasts), which bind together to form a cell pellet. Then, a given preferential direction in the nanoparticle-loaded cell pellet was recorded using an external field of 2 tesla and that the same recorded direction was also read in a 0.5 tesla MRI scanner. After this procedure, map changes in the aggregation of the cells can be detected, such as a disaggregation of the cells induced by the addition of trypsin (Figure 6).

[0075] 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.

[0076] 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.

[0077] The following claims further set out particular embodiments of the disclosure.

Claims

C L A I M S1. A composition comprising an embedding agent and an anisotropic contrast agent for low-field magnetic resonance imaging, wherein said anisotropic contrast agent comprises: an iron oxide nanoparticle; a capping agent, wherein the capping agent is selected from a list comprising dextran, polyethylene glycol), organosilanes, chelates, polymers of silica, and mixtures thereof.

2. The composition according to the previous claim wherein the iron oxide nanoparticle is a doped epsilon iron oxide.

3. The composition according to the previous claim wherein the iron oxide nanoparticle is doped with Al3+or Ga3+.

4. The composition according to any of previous claims 2-3 wherein the molar ratio between the doping ion and iron ranges from 0 to 0.4.

5. The composition according to any of the previous claim wherein the iron oxide is an iron oxide with formula e-GaxFe2-xO3, wherein x ranges from 0 to 0.5.

6. The composition according to any of the previous claims wherein the iron oxide nanoparticle is a ferrite.

7. The composition according to any of the previous claims wherein the concentration of the contrast agent ranges from 0.001 to 10 mg / mL.

8. The composition according to any of the previous claims wherein the size of the largest dimension of the iron oxide nanoparticle ranges from 20 to 40nm, measured by transmission electron microscopy.

9. The composition according to any of the previous claims wherein the capping agent comprises a carboxylic group, an amine groups, a hydroxyl group, a thiol group, or mixtures thereof.

10. Use of a composition as described in any of the previous claims as an anisotropic contrast agent for magnetic resonance imaging.

11. Use of a composition according to any of the previous claims as an enhancer of contrast for magnetic resonance imaging.

12. Use of a composition according to any of the previous claims as an imaging probe, in particular wherein the contribution from the probes is separated from the contribution from the regions where the probes are absent.

13. Hydrogel comprising the contrast agent according to any of the previous claims.

14. Method for obtaining an anisotropic contrast agent for low-field magnetic resonance imaging according to any of the previous claims 1-9, comprising the following steps: incorporation of the contrast agent into a matrix; recording of a suitable direction using an external magnetic field; measuring of an image in two different directions; separating the isotropic and anisotropic components to obtain an image of the distribution of contrast agents in the matrix.

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