Method of incorporation of a metal cation in a material comprising catechol moieties

Abstract

Positron emission tomography (PET) is a bioimaging technique that relies on positron emitting probes. PET probes can be obtained from a non-labeled probe by a late-stage radiolabelling step with a radionuclide, prior to the injection of the probe to the patient. Because of the short half-life duration of most radionuclides, the radiolabelling step must be rapid. The present invention provides a method allowing the rapid embedding of 68Ga into particles comprising polydopamine. The particles labeled with the method are very stable and are obtained in high yield with a high purity. It has been shown that radiolabeled particles obtained with the method of the invention and functionalized with antibodies were efficient in revealing lung and kidney inflammation via a whole-body PET scan in a LPS-induced sepsis mouse model. Such probes, when they further incorporate magnetic nanoparticles, carry a high potential to improve the diagnosis of inflammation via PET-MRI.

Description

[0001] Method of incorporation of a metal cation in a material comprising catechol moieties

[0002] The present invention relates to a method for the incorporation of a metal cation in a material comprising catechol moieties and in particular for the incorporation of gallium into polydopamine (PDA) particles for radiolabeling purposes.

[0003] Nuclear medicine approach involves the use of radioactive substances introduced inside the body to diagnose and cure diseases. An area of research in this field concerns the development of new radiopharmaceuticals, including radiotracers. Radiopharmaceuticals and radiotracers notably comprise organic compound complexed with a metal radionuclide such as201Th,99mTc,67Ga,68Ga,MCu, and89Zr.

[0004] Among the diagnosis techniques used in nuclear medicine, Positron emission tomography (PET) is a common imaging technique. It relies on the emission of positrons by a radiotracer which reacts rapidly with neighboring electrons to emit gamma photons providing a three-dimensional information on the localization of the radiotracer.

[0005] The physical half-life of a radionuclide is an important feature to consider depending on the application. It must be long enough to cover the complexation step needed for obtaining the desired radiopharmaceutical and the administration step. Thus, it is desirable to have efficient and rapid methods for the complexation of the metal radionuclide to the organic compound part. In particular, a promising metal radionuclide for PET,68Ga, has a half-life duration of 68 minutes.

[0006] Previously, Gauberti et al. (W02023007003A1) reported a biocompatible PDA containing magnetic particle suitable for Magnetic Resonance Imaging (MRI) which could be utilized, once radiolabeled with, e.g.,68Ga for PET.

[0007] However, existing methods for the incorporation of gallium generally require, prior to the labeling step, additional conjugation to a DOTA (l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid) like macrocyclic bifunctional chelator. Qiao etal. (Ceramics International 2019, 45, 22183-22195) reported a gallium introduction procedure where Ga(III) was introduced into PDA-functionalized SrTiO; nanotubes. However, in Qiao et al., the complexation of gallium required a reaction time of at least 60 minutes at 100°C and was performed in a Tris buffer at pH = 8.5. Such long reaction times exceed the half-life duration of the68Ga.

[0008] The present invention provides an efficient method allowing the incorporation of gallium into PDA particles, which does not require a prior conjugation of said particles with a bifunctional chelator.

[0009] Surprisingly, it has been found that using a pH of 2 to 5 for the complexation of a metal cation to a material comprising catechol moieties (such as PDA) instead of the basic pH used in the prior art (e.g. in Qiao et al.) allowed a rapid complexation and the obtention of a very stable product in high yields. The present method can also be used for the efficient incorporation of metal cations in other types of material comprising catechol moieties, for example for providing said material with catalytic or antibacterial properties (depending on the metal cation incorporated).

[0010] Description of the figures

[0011] Figure 1 shows Energy-dispersive X-ray Spectroscopy (EDS) mapping and high-angle annular darkfield (HAADF) images of M3P (upper panel) and M3P labeled with Ga3+([natGa]Ga-M3P; lower panel).

[0012] Figure 2 are decay-corrected radiochemical yields of different [68Ga]Ga-MPs.

[0013] Figure 3 are radio-iTLC characterization of [68Ga]Ga-MPs.

[0014] Figure 4 are bar charts showing the in vitro stability at 15 minutes post-incubation of the different radiolabeled [68Ga]Ga-MPs (n = 3).

[0015] Figure 5 displays the dynamic imaging series of immuno-PET acquisitions after the injection of [68Ga]Ga-M3P@aVCAM-l in a healthy mouse vs. sepsis mouse.

[0016] Figure 6 are time activity curves in heart, lungs, kidneys, and liver measured from in vivo PET acquisitions on mice injected with [68Ga]Ga-M3P@aVCAM-l (n = 4 / group).

[0017] Figure 7 are time activity curves in heart, lungs, kidneys, and liver measured from in vivo PET acquisitions on mice injected with [68Ga]Ga-M3P@IgG (n = 4 / group).

[0018] Figure 8 are standard tracer uptake values quantification from in vivo PET acquisitions (n = 4, two-way Anova analysis with Tukey’s multiple comparison test, *p<0.05, **p<0.01, ****p<0.0001).

[0019] Figure 9 are kidney immuno-MRI acquisitions before (baseline scan) and after intravenous injection of [68Ga]Ga-M3P@aVCAM-l in a healthy mouse (top) and a sepsis mouse (bottom). Signal uptake is shown in orange color.

[0020] Figure 10 are kidney mean gray uptake values (n = 4, one-way Anova analysis with Tukey’s multiple comparison tests, *p<0.05, **p<0.01).

[0021] Description

[0022] The present invention provides the following items:

[0023] 1. Item 1: Method of preparation of a material B comprising a metal cation M, said method comprising the following steps: a. Providing a material A, wherein a surface of said material A comprises catechol moieties, b. Providing the metal cation M and contacting it with the material A to obtain the material B, c. Recovering the material B,

[0024] Wherein at step b. the contacting is performed in an aqueous medium W having a pH of 2 to 5.

[0025] As defined herein metal atoms comprise and are limited to alkali metals, alkaline earth metals, lanthanides, actinides, transition metals, and post-transition metals (i.e. Al, Ga, In, Tl, Sn, Pb, and Bi).

[0026] The pH range of step b. is lower than the pKa of catechol, i.e. about 9.45, but still allows complexation with metal cations.

[0027] As defined herein an aqueous medium is a liquid medium wherein water represents at least 50% of the total weight of the solvents comprised in said liquid medium.

[0028] The material A being a material, it should be understood that it does not dissolve in the aqueous medium W.

[0029] The step of providing the material A and the step of providing cation M and contacting it with material A are two distinct steps.

[0030] The material B is the product of the reaction between the metal cation M and the material A. It is believed that said reaction comprises a complexation of the metal cation by the catechol moieties comprised in the material A.

[0031] As defined herein a catechol moiety is a moiety having the following formula:

[0032] 2. Item 2: the method of item 1, wherein the material A is devoid of the metal cation M.

[0033] 3. Item 3: the method of item 1 or 2, wherein the material A is in the form of particles.

[0034] 4. Item 4: the method of item 3, wherein the particles of material A have a hydrodynamic diameter of 100 to 1000 nm.

[0035] According to the present disclosure, the hydrodynamic diameter of a particle (including particles of material A, particles of material B, and magnetic nanoparticles) is measured by dynamic light scattering. The hydrodynamic diameter is defined by the ISO standard 22412:2017.

[0036] 5. Item 5: the method of any one of items 1 to 4, wherein the material B is in the form of particles.

[0037] For obtaining a material B in the form of particles, it is convenient to provide the material A already in the form of particles. 6. Item 6: the method of item 5, wherein the particles of material B have a hydrodynamic diameter of 100 to 1000 nm.

[0038] 7. Item 7 : the method of any one of items 1 to 6, wherein the catechol moieties are moieties in one or more structural units of a polymeric or oligomeric material comprised in material A.

[0039] As defined herein, a polymeric material may be a copolymer.

[0040] A structural unit of a polymeric material or an oligomeric material is a building block of the polymer or oligomer chain. It is the form taken by a monomer after it has been included in the chain.

[0041] Oligomers differ from polymers in that properties of oligomers vary significantly with the removal of one structural unit.

[0042] 8. Item 8: the method of item 7, wherein the polymeric or oligomeric material comprises structural units obtainable by oxidative coupling of a monomer A comprising the following moiety (I):

[0043] As defined herein oxidative coupling is a reaction, where a C-C bond is formed between two carbon atoms that are a-carbons relative to the C-OH and that are not themselves bearing a -OH group:

[0044] In the above reaction scheme, the reactants can be monomers or oligomers or polymers. Said oligomers of polymers may have been formed from the monomer during the oxidative coupling reaction.

[0045] 9. Item 9: the method of item 8, wherein the monomer A has a molar mass of less than 300 g / mol.

[0046] Because the monomer A comprises a catechol moiety, its minimum molar mass is 110.1 g / mol.

[0047] 10. Item 10: the method of item 8 or 9, wherein the monomer A comprises the following moiety

[0048] (II): 11. Item 11 : the method of item 10, wherein the monomer A is dopamine.

[0049] Dopamine has the following formula:

[0050] In the reaction conditions for oxidative coupling other bonds may form or break. For example, when dopamine is used as a monomer further oxidation-cyclization may occur as well as tautomerism on the product of cyclization:

[0051] 12. Item 12: the method of any one of items 1 to 11, wherein the material A is a magnetic material.

[0052] As defined herein, a magnetic material is a material that is attracted to a magnet. A magnetic material can be, for example, a ferrimagnetic oxide or a ferromagnetic oxide. An advantage of using a magnetic material in the form of particles is that it allows to use a magnetic separation step to purify the particles. This provides in particular a rapid and efficient means for purifying the magnetic material or changing the medium in which it is dispersed. This rapidity and efficiency are crucial when the material is to be used as a radiopharmaceutical and has a relatively short half-life. Using a magnetic material is also advantageous for the use of the material B as an MRI contrast agent as magnetic materials provide enhanced contrast in MRI.

[0053] 13. Item 13: the method of any one of items 1 to 12, wherein the material B is a magnetic material.

[0054] For obtaining a magnetic material B, it is convenient to provide a material A that is already a magnetic material.

[0055] 14. Item 14: the method of item 12 or 13, wherein the material A comprises magnetic nanoparticles A in a matrix of the polymeric or oligomeric material.

[0056] When the material A is in the form of particles, said particles contain the magnetic nanoparticles in a matrix of the polymeric or oligomeric material.

[0057] 15. Item 15: the method of item 14, wherein the magnetic nanoparticles represent 30 to 95 % w / w, preferably 40 to 85 % w / w of the total mass of material A.

[0058] 16. Item 16: the method of item 14 or 15, wherein the hydrodynamic diameter of the magnetic nanoparticles in material A is of 5 nm to 100 nm. 17. Item 17: the method of any one of items 14 to 16, wherein the magnetic nanoparticles in material A are metal oxide nanoparticles.

[0059] 18. Item 18: the method of item 17, wherein the metal oxide is an iron oxide.

[0060] 19. Item 19: the method of item 18, wherein the iron oxide is an oxide of iron (II) and iron (III).

[0061] 20. Item 20: the method of any one of items 14 to 19, wherein the material B comprises the magnetic nanoparticles A.

[0062] 21. Item 21: the method of any one of items 1 to 20, wherein the material A comprises a targeting moiety A able to bind a molecular target.

[0063] 22. Item 22: the method of item 21, wherein the targeting moiety A is an antibody.

[0064] Notably, with the method of the invention, the complexation of the metal cation is possible even when the material A comprises antibodies and the targeting properties of said antibodies are retained in material B.

[0065] 23. Item 23: the method of item 21 or 22, wherein the targeting moiety A is covalently linked to the polymeric or oligomeric material.

[0066] 24. Item 24: the method of any one of items 21 to 23, wherein the material B comprises the targeting moiety A.

[0067] 25. Item 25: the method of any one of items 1 to 24, wherein the metal cation M is a metal cation of group 13 of the periodic table.

[0068] 26. Item 26: the method of item 25, wherein the metal cation is Ga3+.

[0069] The metal cation may be a radioactive isotope, to provide the material B with specific properties. Preferably, the metal cation is a positron emitting radioisotope, such as68Ga3+. In this case, it can be used for PET, preferably in this case, the material B is in the form of particles.

[0070] 27. Item 27 : the method of any one of items 8 to 26, wherein step a. of providing the material A comprises the following substeps: i. Providing the monomer A, ii. In an aqueous medium W 1 , contacting the monomer A with a base to induce oxidative coupling of the monomer A, iii. Recovering the material A comprising the reaction product of the oxidative coupling of the monomer A.

[0071] When monomer A is dopamine, it may be provided as a salt thereof, e.g. a hydrochloride thereof.

[0072] 28. Item 28: the method of item 27, wherein substep a.ii. is performed under stirring.

[0073] When stirring is performed during substep a.ii., material A is obtained in the form of particles. 29. Item 29: the method of item 27 or 28, wherein the amount of base is of 1 to 20 equivalents per mole of monomer A.

[0074] Equivalents represent a molar amount of functionalities of the base that will be able to react to induce oxidative coupling. For example, when the base is ammonia, there is one such functionality per ammonia molecule.

[0075] 30. Item 30: the method of any one of items 27 to 29, wherein the base is a Brpnsted base.

[0076] 31. Item 31 : the method of item 30, wherein the base is an amine.

[0077] As defined herein an amine is a compound containing a basic nitrogen atom with a lone pair. As defined herein amines include ammonia.

[0078] 32. Item 32: the method of item 31 , wherein the base is ammonia.

[0079] 33. Item 33: the method of any one of items 27 to 32, wherein the aqueous medium W1 comprises 10% w / w to 50% w / w of ethanol relative to the total mass of the solvents contained in the aqueous medium W 1 , preferably wherein the rest of the solvents in the aqueous medium W 1 is water.

[0080] 34. Item 34: the method of any one of items 27 to 32, wherein the aqueous medium W1 comprises at least 90% w / w of water relative to the total mass of the solvents contained therein, preferably wherein water is the only solvent in the aqueous medium W 1.

[0081] 35. Item 35: the method of any one of items 27 to 34, wherein, at substep a.i., the monomer A is provided in mixture with the magnetic nanoparticles A.

[0082] 36. Item 36: the method of any one of items 1 to 35, wherein the aqueous medium W has a pH of 2.5 to 4.5.

[0083] 37. Item 37: the method of any one of items 1 to 36, wherein the aqueous medium W comprises at least 90% w / w of water relative to the total mass of the solvents contained therein, preferably wherein water is the only solvent in the aqueous medium W.

[0084] 38. Item 38: the method of any one of items 1 to 37, wherein the aqueous medium W comprises an additive A dissolved therein, wherein said additive A has a molar mass of less than 400 g / mol and comprises the following moiety (IV):

[0085] It is believed that, in the method of the inventions, the presence of the additive comprising the moiety IV provides a weakly complexing environment for the cation M, which ensures a good supply of dissolved cation M for complexation with the more strongly complexing material A. In the absence of an additive comprising the moiety IV, the product obtained immediately after step b of the method may have a lower purity. 39. Item 39: the method of item 38, wherein the additive A comprises the following moiety (V):

[0086] 40. Item 40: the method of item 38 or 39, wherein the additive A is a buffering agent.

[0087] 41. Item 41: the method of any one of items 38 to 40, wherein the additive A comprises at least one sulfo group.

[0088] A sulfo group is a moiety of formula:

[0089] H o-°H

[0090] 42. Item 41: the method of item 41, wherein the additive A is HEPES.

[0091] HEPES is (4-(2-hydroxyethyl)-l -piperazineethanesulfonic acid):

[0092] 43. Item 43: the method of any one of items 38 to 43, wherein the aqueous medium W comprises between 0.015 mol / L and 2 mol / L of additive A.

[0093] 44. Item 44: the method of any one of items 1 to 43, wherein step b. is performed at a temperature comprised between 10 and 35°C.

[0094] 45. Item 45: the method of any one of items 1 to 44, wherein at step b., contacting the metal cation M with the material A is performed for a duration of 1 to 15 minutes, preferably of 2 to 10 minutes.

[0095] The duration of the contacting the metal cation M with the material A starts at the time when the metal cation M and the material A are first contacted and ends at the time when the material B is recovered, e.g. by separation from the aqueous medium W.

[0096] 46. Item 46: the method of any one of items 1 to 45, wherein at step b., the metal cation M is dissolved in the aqueous medium W.

[0097] 47. Item 47: the method of item 46, wherein , at step a., the material A is provided as a suspension A of particles of the material A in an aqueous medium A and wherein, at step b., an aqueous solution B of the metal cation M is added to the suspension A to perform the contacting of the material A with the metal cation M in the aqueous medium W, which results from the addition of the solution B to the aqueous medium A.

[0098] 48. Item 48: the method of item 47, wherein the aqueous medium A comprises the additive A. 49. Item 49: the method of item 47 or 48, wherein the aqueous medium A has a pH comprised between 2 and 7, preferably between 3 and 5.

[0099] 50. Item 50: the method of any one of items 47 to 49, wherein solution B has a pH of below 7.

[0100] 51. Item 51: the method of any one of items 47 to 50, wherein solution B is a68Ga3+solution obtained by elution from a68Ge / 68Ga generator.

[0101] Examples

[0102] The experimental protocols below describe the preparation of particles comprising PDA, their labeling with68Ga according to the method of the present invention, and the use of the obtained particles as bimodal PET-MRI probe.

[0103] Two types of particles were prepared: particles comprising only PDA and particles comprising iron oxide magnetic nanoparticles (ION) in a matrix of PDA (hereinafter called M3P for “microsized matrixbased magnetic particles”).

[0104] The M3P were functionalized with a rat anti-mouse VCAM-1 antibody (M3P@aVCAM-l) and to the matching isotype control (M3P@IgG) for the imaging experiments. The attachment of the antibodies relies on Schiff base reactions or Michael additions via thiol or primary amine groups from the immunoglobulin to the PDA matrix.

[0105] By incubating M3P at room temperature for 10 minutes in an aqueous solution of GaCE, [natGa]Ga-M3P were formed. Transmission electronic microscopy and high-angle annular dark-field images confirmed that the structure of the iron oxide clusters remains unchanged while the X-ray spectroscopy analysis confirmed that gallium could be incorporated into the particles (Fig. 1).

[0106] PDA particles, M3P@aVCAM-l and M3P@IgG were also labeled by suspending them in HEPES buffer (0.3 M, pH 4) and contacting them with a68Ga solution in HC1 (0.1 M) and stirring the mixture for 5 minutes at room temperature and the reactions were controlled by radio-ITLC by using citrate buffer (0.1 M, pH 4.9) as eluant.

[0107] Using these conditions, all68Ga labelled microparticles ([68Ga]Ga-MPs) are found at the baseline of the radio-iTLC (Rf = 0 - 0.2), while free68Ga3+present as [68Ga]Ga-citrate migrates to the solvent front (Rf = 1.0). After magnetic purification to discard uncomplexed68Ga3+and resuspension in mannitol (0.3M), all [68Ga]Ga-MPs were obtained in quantitative radiochemical yield (RCYs >98%, Fig. 2) and high radiochemical purity (RCPs >99%, Fig. 3).

[0108] When an acetate buffer (1.5 M, pH 4) was used instead of the HEPES buffer, the product directly had a lower RCP than with HEPES resulting in an overall lower RCY and a slightly lower RCP after magnetic purification. To further validate the use of [68Ga]Ga-M3P@mAbs as immuno-PET-MR contrast agents in vivo multiple in vitro challenge experiments were performed (Fig. 4). First the stability towards ligand exchange was carried out using H4EDTA as a competitor. Briefly, [68Ga]Ga-MPs were incubated with 1000 equivalents of EDTA (0.1 M in chelex-treated water, pH 7.4) and gently stirred for 15 minutes. Under these conditions all the [68Ga]Ga-MPs remained stable. When incubated with either transferrin or Fe(III) as competitor the stability of [68Ga]Ga-M3P was slightly lower compared to [68Ga]Ga-PDA but remained high at 88.9 ± 6.9 and 86.8 ± 6.8, respectively (the numbers refer to the percentage of the counted radioactivity at Rf = 0 on the TLC plate). Finally, the stability of the radiolabelled MPs in human serum (a more biological relevant set up) was investigated. After incubation in human serum the different [68Ga]Ga-MPs showed full stability thus allowing their further in vivo evaluation as bimodal PET-MRI tracers.

[0109] The bimodal probe was tested in a preclinical positron emission tomography (PET) coupled to a 7 Tesla magnetic resonance imaging (MRI) system (7T PET / MRI, Brucker), in a mouse model of sepsis induced by intraperitoneal injection of lipopolysaccharide (LPS, 5mg / kg). LPS is an outer membrane component of gram-negative bacteria that induces an innate immune response via stimulation of toll-like receptor 4. This model is characterized by strong systemic inflammation and multiple organ dysfunction, with the lung, the heart, and the kidneys being the most vulnerable and critical.

[0110] The pharmacokinetic profile of the particles radiolabeled with68Ga and functionalized with anti-VCAM- 1 antibody ([68Ga]Ga-M3P@aVCAM-l) was monitored via whole-body dynamic PET scan after intravenous injection to healthy and sepsis mice (Fig. 5). The signal accumulated during the 15 seconds post-injection thus displays the blood pool. In the healthy animals, a fast accumulation in the liver is observed (Fig. 6), which is in agreement with the extremely short half-life of these particles previously observed due to the fast sequestration by the mononuclear phagocyte system. In the sepsis mice, a strong signal was detected in the lung and in the kidneys (Fig. 6) indicating that the particles rapidly accumulated in those two specific tissues.

[0111] When injected with particles functionalized with the matching isotype control antibody ([68Ga]Ga- M3P@IgG), no signal uptake other than the liver was noted in both sepsis and healthy mice (Fig. 7), indicating that the signal observed in the sepsis cohort with the [68Ga]Ga-M3P@aVCAM-l is specific for VCAM-1 expression.

[0112] PET signal was measured within volume of interest drawn from MRI scan on tissues of interest and presented as mean standard uptake values (SUVmean) (Fig. 8). The analysis revealed a significant uptake within the brain, the heart, the kidneys, and the lungs of the sepsis mice injected with VCAM-1 targeted particles compare to the other groups. This indicates strong inflammation and upregulation of VCAM- 1 in those tissues. The kidney conditions via examined more precisely via immuno-MRI (Fig. 9). The iron oxide content of the [68Ga]Ga-M3P@aVCAM-l provided a sensitive contrast in T2*-weighted acquisition and enabled high resolution mapping of VCAM-1 expression within the kidneys. Signal uptake compared to baseline pre-injection scan was measured and was found significantly higher in the kidneys from the sepsis animals injected with [68Ga]Ga-M3P@aVCAM-l compared to the signal uptake obtained in the kidneys from the healthy animals injected with the same particles from the sepsis animals injected with the non-targeted control particles [68Ga]Ga-M3P@IgG (Fig. 10). We pooled the signal obtained via immuno-PET and immuno-MRI in the kidneys from the different animals for which both were performed and studied the relationship. We measured a strong positive and significant Pearson correlation (r = 0.787, p<0.01) and a linear regression with a good coefficient of determination (R2= 0.619) confirming that both immuno-imaging modalities and the image analysis methods provide reliable and consistent measures of the signal associated to the contrast agent. Histological analysis also confirmed that the particles were localized in kidneys from a sepsis animal in the vessel area characterized by VCAM-1 expression, but not in the kidneys from a healthy animal.

[0113] The labelling method of the invention thus provides rapid and strong complexation of68Ga to PDA whether alone or in the presence of iron oxide nanoparticles or in the presence of antibodies. The labelled particles retain the functionalities imparted by the magnetic iron oxide or by the antibodies after complexation and are sufficiently rapidly obtained and stable enough to be used as bimodal PET-MRI probe.

[0114] Synthesis of bimodal PET-MRI probe

[0115] Synthesis of microsized matrix-based magnetic particles (M3P)

[0116] Iron oxide nanoparticles (ION) were obtained via co-precipitation method adding 6.3 mL of 13% ammonia progressively to a 5.7 mL aqueous solution of FeC13-6H2O (104.63 mg / mL, Sigma-Aldrich) and FcCL. HiO (142.26 mg / mL, Sigma Aldrich) dissolved in distilled water. The ION were then washed 3 times with distilled water via magnetic separation and resuspended in 40 mL of an aqueous solution of dopamine hydrochloride (2.5 mg / mL, Sigma Aldrich). The ION were then self-assembled into microsized clusters via polymerization of the dopamine induced by the addition of 270 pL of 13% ammonia under continuous stirring for 2 hours using an Ultra-TurraxT-25 disperser at 20,500 rpm. The suspension was then centrifuged at 1000g to remove the large aggregates, the pellet was discarded and the supernatant was washed 3 times with distilled water via magnetic separation and finally resuspended in 8 mL H2O. The black suspension obtained corresponds to the microsized matrix-based magnetic particles (M3P). Particle suspensions presenting a mean hydrodynamic diameter of 396 + 44 nm were obtained. Functionalisation of MSP particles with antibodies:

[0117] M3P suspension (1 mL) was incubated with 500 pg of anti-VCAM-1 antibody (M3P@aVCAM-l, A(429), BD Biosciences) or 500 pg match isotype polyclonal IgG (M3P@IgG, Sigma Aldrich) into 5 mL phosphate buffer (10 mM, pH=8.5) for 24 hours at room temperature under continuous mild agitation. The obtained solution was sonicated for 40 seconds at 20% amplitude and 26 kHz using a UP200ST sonicator tip to break aggregates, washed 3 times with an aqueous mannitol (0.3 M) solution via magnetic separation, and finally resuspended into 5 mL of mannitol (0.3 M).

[0118] Synthesis of PDA micro-sized particles:

[0119] The micro-sized particles solely composed of PDA, 15 mg of dopamine hydrochloride was added to a 10 mL solution of distilled water with 30% (v / v) ethanol and 0.2 % ammoniac, gently mixed for 6 hours at room temperature.

[0120] Radiolabelling procedure:

[0121] M3P@aVCAM-l, M3P@IgG and PDA particles were first resuspended in 1.5 mL of 0.3M HEPES buffer (pH 4). Then, Gallium-68 (500-1000 MBq in 1.1 mL HC10. IN) eluted from a68Ge / 68Ga generator (IRE Elit, Belgium) was directly added to the previous suspension and allowed to incubate for 5 min at RT. Before further use in vivo, M3P@aVCAM-l and M3P@IgG were purified via magnetic separation and resuspended in 2 mL mannitol 0.3M.

[0122] Measurement methods

[0123] Hydrodynamic diameter measurement:

[0124] Dynamic light scattering was used to determine the average hydrodynamic diameter, the polydispersity index and the diameter distribution by intensity of the M3P particles with a Nano ZS apparatus (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser at a fixed scattering angle of 173°. The temperature of the cell was kept constant at 25 °C, and all dilutions were performed in pure water. Measurements were performed in triplicate.

[0125] EDS Spectroscopy, TEM, HAADF:

[0126] Transmission Electron Microscopy (TEM) was performed by a Tecnai F20 instrument (200 kV). Energy- dispersive X-ray Spectroscopy (EDS) mapping was measured by Hitachi SU7000 FE-SEM and Tecnai F20 instrument, respectively. Nanoparticle suspensions were pipetted on and air-dried on plasma- cleaned Formavar carbon-coated copper grids before TEM measurement.

[0127] Thin layer chromatography: Glass-fibre iTLC plates impregnated with silica-gel (iTLC-SG, Agilent Technologies) were developed by using an aqueous mobile phase containing citrate buffer (0.1M, pH 4.9), and were analyzed on an Elysia Raytest Rita Star 2018203 plate reader (Elysia-raytest GmbH, Straubenhardt, Germany). When using aqueous mobile phases containing citrate buffer (0.1 M, pH 4.9), radiochemical conversion (RCC) was determined by integrating the data obtained by the radio-TLC plate reader and determining both the percentage of radiolabelled product (Rf = 0.0) and ‘free’68Ga (Rf = 1.0; present in the analysis as [68Ga][Ga(citrate)]). Integration and data analysis were performed by using Gina star TLC software. Appropriate background and decay corrections were applied as necessary. The radiolabeling stability of [68Ga]Ga-M3P@aVCAM-l, [68Ga]Ga-M3P@IgG, and [68Ga]Ga-PDA particles were measured in vitro in different conditions.

[0128] Stability tests

[0129] Human serum challenge:

[0130] For each experiment ~2.5 MBq (50 pL) of the different [68Ga]Ga-particles were mixed in 200 pL of human serum. The samples were incubated at 37°C for 2h. The dissociation of the radiolabeled particles was monitored by radio-iTLC (0.1M aqueous citrate at pH 4.9). Experiments were performed in triplicate.

[0131] Transferrin challenge:

[0132] For each experiment ~2.5 MBq (50 pL) of the different [68Ga]Ga particles were mixed with 10 pL of an aqueous Transferrin solution (10 mg / mL). The samples were incubated at 37°C for 2h. The dissociation of the radiolabelled particles was monitored by radio-iTLC (0.1M aqueous citrate at pH 4.9). Experiments were performed in triplicate.

[0133] H4EDTA stability measurements:

[0134] For each experiment ~2.5 MBq (50 pL) of the different [68Ga]Ga-particles were diluted in Chelex- treated water (50 pL) at pH 7-7.4 followed by the addition of 1000 equivalents (based on iron content) H4EDTA (1 mM, pH 7.4). The samples were incubated at 25°C for 2h. The dissociation of the radiolabelled particles was monitored by radio-iTLC (0.1M aqueous citrate buffer at pH 4.9). Experiments were performed in triplicate.

[0135] Iron( III) challenge:

[0136] For each experiment ~ 2.5 MBq (50 pL) of the different [68Ga]Ga-particles were diluted in Chelex- treated water (50 pL) at pH 7-7.4 followed by the addition of 1 equivalent (based on iron content) FcCI ; (100 mg / mL, 10 pL, pH 7.4). The samples were incubated at 25°C for 2h. The dissociation of the radiolabelled particles was monitored by radio-iTLC (0.1M aqueous citrate buffer at pH 4.9). Experiments were performed in triplicate.

[0137] [68Ga]Ga-M3P and [68Ga]Ga-PDA present excellent stability in all these different challenges proving the high stability of the particles of the invention and their suitability for in vivo uses.

[0138] Imaging experiments

[0139] Animal models:

[0140] All experiments were performed on 8- to 10-week-old male Swiss mice (Janvier, France) maintained under specific pathogen-free conditions at the Centre Universitaire de Ressources Biologiques (CURB, Basse-Normandie, France), having free access to food and tap water. Experiments were approved by the local ethical committee of Normandy (CENOMEXA, APAFIS#22318). Sepsis model was induced by a single intraperitoneal injection of LPS (5 mg / kg, Lipopolysaccharides from Escherichia coli O111:B4, Sigma Aldrich) in the lower right quadrant of the abdomen of the animal. The intraperitoneal injection of LPS is followed by a subcutaneous injection of buprenorphine (O.lmg / kg) in order to anticipate any pain or suffering from the animal as soon as possible.

[0141] PET-MRI experiments:

[0142] Experiments were carried out on a small animal 7T PET-MRI system (Brucker, Germany). Mice were anesthetized with isoflurane (1.5 to 2.0%) and maintained at 37°C by the integrated heat animal holder, and the breathing rate was monitored during the imaging procedure. A catheter was inserted into the tail vein of mice for intravenous administration of the contrast agent. For anatomical MRI reference, Tl_Fisp_3D scans were performed including 3 stitched volumes, allowing to obtain a whole body image of a mice, with the following parameters: 3D, repetition time (TR) 5.5 ms, echo time (TE) 2.6 ms, number of averages (NA) 3, voxel spacing 0.5 / 0.5 / 0.5 mm, and a field of view (FOV) 40 / 40 / 108 mm. High-resolution T2*-weighted images of kidneys for immuno-MRI were acquired with a surface coil (Brucker, Germany), using a 3D FLASH gradient echo imaging (spatial resolution of 78 pm by 156 pm by 300 pm) with TE / TR 8.6 ms / 50 ms, and a flip angle of 20°. One baseline scan was performed before the injection of the particles and one after the PET acquisition. List mode PET data were acquired for 10 min, and this was initiated as soon as the formulated [68Ga]Ga-M3P@VCAM-l or [68Ga]Ga- M3P@IgG were injected in order to monitor the bolus. A second similar T2*-weighted images of kidneys was finally acquired to detect the magnetic particles accumulated 10 minutes after injection.

[0143] Image analysis:

[0144] Immuno-PET image data underwent normalization to address discrepancies in PET response, including factors like attenuation, random events, dead-time count losses, positron branching ratio, and physical decay from the time of injection. The Dynamic PET images were reconstructed using an iterative MAP 0.5 algorithm and segmented into 8 different frames (4x30 s; 3 x60 s; 1 x300 s). To standardize the images in terms of %ID cm3(equivalent to %ID / g assuming tissue density as unity), the resulting image data were normalized against the administered activity. Analysis employed PMOD 3.7 software (PMOD, Zurich, Switzerland). Quantification and the creation of time-activity curves (TACs) involved manually drawing 3D volumes-of-interest (VOIs) to ascertain maximum and average radioactivity accumulation (measured in %ID cm3and decay-corrected to the time of injection) across various tissues. Finally, data were transposed into standardized uptake values and were presented as mean values (SUVmean) + SD.

[0145] For kidney MRI image data, analysis were performed with ImageJ software (National Institute of Health). Regions of interest were drawn around the kidney’s areas and the mean gray value was measured in the pre-injection baseline scan and in the post-injection scan. The mean gray uptake (MGU) was calculated from the difference between the gray value measured on the baseline and on the postinjection scan. Data were presented as mean values + SD. The immuno-MRI images were obtained from subtraction of post-injection image to the pre-injection baseline image via the image calculator tool, and the lookup table was changed for fire.

[0146] Statistical analysis:

[0147] Results are presented as mean values + SD. Statistical analysis were performed with Graph Pad Prism software (v8.0). Tissues' SUVmean values measured on immuno-PET acquisitions were compare with two-way Anova followed by Tukey’s multiple comparisons tests. Kidneys’ Mean Gray Uptakes measured on immuno-MRI acquisitions were compared with ordinary one-way Anova followed by Tukey’s multiple comparisons. The numbers of particles functionalized with anti-VCAM-1 antibody versus IgG isotype control in histology sections were compared via a Student’s T test, p < 0.05 was considered significant (two-sided). The correlation between PET and MRI signals in the same animals was studied with a Pearson correlation test. A strong positive and significant Pearson correlation (r = 0.787, p<0.01) and a linear regression with a good coefficient of determination (R2= 0.619) were measured confirming that both immuno-imaging modalities and the image analysis methods provide reliable and consistent measures of the signal associated to the contrast agent.

Claims

Claims1. Method of preparation of a material B comprising a metal cation M, said method comprising the following steps: a. Providing a material A, wherein a surface of said material A comprises catechol moieties, b. Providing the metal cation M and contacting it with the material A to obtain the material B, c. Recovering the material B,Wherein at step b. the contacting is performed in an aqueous medium W having a pH of 2 to 5.

2. The method of claim 1 , wherein the material A is devoid of the metal cation M.

3. The method of claim 1 or 2, wherein the material A is in the form of particles.

4. The method of any one of claims 1 to 3, wherein the catechol moieties are moieties in one or more structural units of a polymeric or oligomeric material comprised in material A.

5. The method of claim 4, wherein the polymeric or oligomeric material comprises structural units obtainable by oxidative coupling of a monomer A comprising the following moiety (I):T T6. The method of claim 5, wherein the monomer A comprises the following moiety (II):

7. The method of claim 6, wherein the monomer A is dopamine.

8. The method of any one of claims 1 to 7, wherein the metal cation M is a metal cation of group13 of the periodic table.

9. The method of claim 8, wherein the metal cation is Ga3+.

10. The method of any one of claims 5 to 9, wherein step a. of providing the material A comprises the following substeps: i. Providing the monomer A, ii. In an aqueous medium W 1 , contacting the monomer A with a base to induce oxidative coupling of the monomer A, iii. Recovering the material A comprising the reaction product of the oxidative coupling of the monomer A.

11. The method of any one of claims 1 to 10, wherein the aqueous medium W comprises an additive A dissolved therein, wherein said additive A has a molar mass of less than 400 g / mol and comprises the following moiety (IV):

12. The method of claim 11, wherein the additive A is HEPES.

13. The method of any one of claims 1 to 12, wherein step b. is performed at a temperature comprised between 10 °C and 35 °C.

14. The method of any one of claims 3 to 13, wherein, at step a., the material A is provided as a suspension A of particles of the material A in an aqueous medium A and wherein, at step b., an aqueous solution B of the metal cation M is added to the suspension A to perform the contacting of the material A with the metal cation M in the aqueous medium W, which results from the addition of the solution B to the aqueous medium A.

15. The method of claim 14, wherein solution B is a68Ga3+solution obtained by elution from a 68Ge / 68Ga generator.

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