Conjugate comprising self-assembled biocompatible particles and an activator of fibrinolysis for use in the treatment of a condition requiring thrombolysis

Abstract

The inventors have now succeeded in efficiently conjugating clusters of ultrasmall particles of iron oxide (USPIO) and polycathecolamine or polyserotonine with an activator of fibrinolysis, in particular a tissue plasminogen activator (tPA). Such conjugates are useful for detecting microthrombi in a condition requiring thrombolysis, in particular acute ischemic stroke, by magnetic resonance imaging (MRI), and for treating a condition requiring thrombolysis, in particular acute ischemic stroke. The present invention relates to conjugate comprising a suspension of biocompatible particles comprising ultrasmall particles of iron oxide embedded in a polycathecolamine or polyserotonine matrix, an activator of fibrinolysis or a fragment thereof and optionally a deoxyribonuclease, and its use in an in vivo method of detection of microthrombi in a condition requiring thrombolysis, in particular acute ischemic stroke, by magnetic resonance imaging (MRI) and in the treatment of a condition requiring thrombolysis, in particular ischemic stroke.

Description

[0001] CONJUGATE COMPRISING SELF-ASSEMBLED BIOCOMPATIBLE PARTICLES AND AN ACTIVATOR OF FIBRINOLYSIS FOR USE IN THE TREATMENT OF A CONDITION REQUIRING THROMBOLYSIS

[0002] The present invention relates to a conjugate comprising a suspension of biocompatible particles comprising ultrasmall particles of iron oxide embedded in a polycathecolamine or polyserotonine matrix, an activator of fibrinolysis or a fragment thereof and optionally a deoxyribonuclease, and its use in an in vivo method of detection of microthrombi in a condition requiring thrombolysis, in particular acute ischemic stroke by magnetic resonance imaging (MRI) and in the treatment of a condition requiring thrombolysis, in particular ischemic stroke.

[0003] BACKGROUND OF THE INVENTION

[0004] Ischemic stroke (IS) treatments permit reperfusion of the occluded vessel through thrombolysis, combined or not with thrombectomy. Unfortunately, even if the recanalization is complete, the microcirculation downstream remains obstruct by microthrombi (Bathia, R. et al., Stroke 2010, 41, 2254-2258). The origin of microthrombi can be either through the disintegration of the proximal clot or the formation of microvascular thrombosis (Goyal, M. et al., Stroke 2021, 52, 1147-1153). The presence of microthrombi is associated with a worse functional prognosis. Notably, it has been demonstrated that patients who exhibit microthrombi after successful reperfusion of arteries are at risk of experiencing cognitive decline and dementia (Goldberg, I. et al., J. Neurol. Sci. 2012, 322, 250-253). The animal model of ischemic stroke in mice developed by thrombin injection into the middle cerebral artery generates microthrombi in the ischemic cortex (Orset, C. etal., Stroke 2017, 38, 2271- 2278).

[0005] The aim of current treatments for ischemic stroke is reperfusion of the occluded vessel through thrombolysis, combined or not with thrombectomy. However, even when a full recanalisation is achieved, a lack of reperfusion in the downstream vascularisation is often observed. This is correlated with the presence of microthrombi in the vessels of the infarcted area. Microthrombi are suspected to contribute to dementia and cognitive decline and are currently misdiagnosed due to the absence of precise methods. Thus, there is a need for a method for detecting and treating microthrombi during acute ischemic stroke in magnetic resonance imaging (MRI).

[0006] The inventors have previously developed a new type of contrast agent made of selfassembled submicrometric clusters of ultrasmall particles of iron oxide (USPIO) using a polycathecolamine or polyserotonine, in particular poly dopamine (PDA), as an embedding matrix. Using only three reagents (iron chloride, a polycathecolamine or polyserotonine, and ammonia), submicrometric clusters of USPIO and polycathecolamine or polyserotonine with mean diameters ranging from 250 nm to 900 nm were produced. Thanks to the biocompatible, hydrophilic and reactive coating of polycathecolamine or polyserotonine (Lee, H. et al., Science 2007, 318, 426-430; Wu, D. et al., Chem. Soc. Rev. 2021, 50, 4432- 4483), the clusters of USPIO and polycathecolamine or polyserotonine can be efficiently conjugated with targeting moieties such as proteins.

[0007] SUMMARY OF THE INVENTION

[0008] The inventors have now succeeded in efficiently conjugating such clusters of USPIO and polycathecolamine or polyserotonine with an activator of fibrinolysis, in particular a tissue plasminogen activator (tPA) and optionally a deoxyribonuclease. Such conjugates are useful for detecting microthrombi in a condition requiring thrombolysis, in particular acute ischemic stroke, by magnetic resonance imaging (MRI) and for treating a condition requiring thrombolysis, in particular acute ischemic stroke.

[0009] The present invention therefore provides a conjugate comprising a suspension of particles and an activator of fibrinolysis or a fragment thereof, wherein the suspension of particles comprises particles having a hydrodynamic diameter comprised between 200 and 2000 nm, said particles comprising ultrasmall particles of iron oxide having a diameter between 1 and 50 nm embedded within a polymer matrix selected from polycathecolamines or polyserotonine.

[0010] The invention also relates to a suspension comprising the conjugate of the invention and a physiological medium. The invention also relates to the conjugate of the invention or the suspension of the invention, for use in an in vivo method of detection of microthrombi in a condition requiring thrombolysis, comprising the steps of: a) administering to a patient the conjugate of the invention or the suspension of the invention; b) detecting the microthrombi by magnetic resonance imaging (MRI).

[0011] The invention also relates to the conjugate of the invention or the suspension of the invention, for use in the treatment of a condition requiring thrombolysis.

[0012] DETAILED DESCRIPTION OF THE INVENTION

[0013] As detailed above, the invention relates to a conjugate comprising a suspension of particles and an activator of fibrinolysis or a fragment thereof, wherein the suspension of particles comprises particles having a hydrodynamic diameter comprised between 200 and 2000 nm, preferably between 250 and 1250 nm, more preferably between 300 and 1000 nm, still more preferably between 500 and 1000 nm, said particles comprising ultrasmall particles of iron oxide having a diameter between 1 and 50 nm embedded within a polymer matrix selected from polycathecolamines or polyserotonine.

[0014] In the context of the invention, the term “conjugate” refers to a molecule composed of two or more molecules, in particular two molecules, which are linked together. The conjugates of the invention are typically composed of particles as defined above linked to a molecule comprising free amine or thiol groups, such as a protein, in particular protein that can be a tissue plasminogen activator (tPA) or a fragment thereof.

[0015] In other words, the invention relates to a suspension of particles conjugated to an activator of fibrinolysis or a fragement thereof, wherein the particles have a hydrodynamic diameter comprised between 200 and 2000 nm, preferably between 250 and 1250 nm, more preferably between 300 and 1000 nm, still more preferably between 500 and 1000 nm and comprises particles of iron oxide having a diameter between 1 and 50 nm embedded within a polymer matrix selected from polycathecolamines or polyserotonine. As used herein, the expression “comprised between ... and ...” should be understood to include the boundaries of the recited range.

[0016] As used herein, the term “embedded” relative to nanoparticles and the surface of the polymer matrix refers to the nanaoparticles being at least partially extended into the surface such that the polymer is in contact with the nanoparticle to a greater degree than would accur if the nanoparticles were simply laid on the surface of the polymer matrix.

[0017] In the context of the invention, the term “hydrodynamic diameter” refers to the diameter of a hypothetical hard sphere that diffuses with the same speed as the particle being measured. It reflects the size of the particle when in solution and includes coatings or surface modifications made to the particle in question.

[0018] The hydrodynamic diameter of the particles of the suspension of particles comprised in the conjugate of the invention may be determined according to any method known by the person skilled in the art. In particular, the hydrodynamic diameter of the particles may be determined by dynamic light scattering (DLS), with for example a NanoZS® apparatus (Malvern Instruments, Worcestershire, UK) equipped with a 633 nm laser at a fixed scattering angle of 173°, with the temperature of the cell being kept constant at 25°C. The particles are for this measure put in suspension in water at a concentration of 20 pg to 200 pg of iron per mL of water. Other known methods are particle tracking analysis (PTA) or its variant nanoparticle tracking analysis (PTA).

[0019] For example, the hydrodynamic diameter of the particles may be comprised between 200 and 2000 nm, between 200 and 1250 nm, between 200 and 1000 nm, between 200 and 900 nm, between 200 and 800 nm, between 200 and 700 nm, between 250 and 2000 nm, between 250 and 1250 nm, between 250 and 1000 nm, between 250 and 900 nm, between 250 and 800 nm, between 250 and 700 nm, between 300 and 2000 nm, between 300 and 1250 nm, between 300 and 1000 nm, between 300 and 900 nm, between 300 and 800 nm, between 300 and 700 nm, between 400 and 2000 nm, between 400 and 1250 nm, between 400 and 1000 nm, between 400 and 900 nm, between 400 and 800 nm, between 400 and 700 nm, between 500 and 2000 nm, between 500 and 1250 nm, between 500 and 1000 nm, between 500 and 900 nm, between 500 and 800 nm or between 500 and 700 nm. Ultrasmall particles of iron oxide incorporated in the particles of the invention can be chosen among maghemite of formula FC2O3, magnetite of formula FC3O4 or a mixture of FC2O3 and FeaC . These different types of iron oxide are all superparamagnetic and biocompatible, allowing their use in particular as contrast agents in Magnetic Resonance Imaging (MRI) or as tracers in Magnetic Particle Imaging (MPI). Accordingly, the iron oxide of the ultrasmall particles of iron oxide comprised in the particles comprised in the suspension of particles of the conjugate of the invention may be selected tion FC2O3, FC3O4, or a mixture of FC2O3 and FC3O4.

[0020] As used herein, the term “nanoparticle” refers to a material having a particle size of less than 1000 nm, in particular ranging from 1 to 500 nm, more particularly from 1 to 300 nm.

[0021] As used herein, the term “ultrasmall particle” refers to a material having a particle size of less than 50 nm, in particular ranging from 1 to 50 nm, more particularly from 1 to 30 nm.

[0022] In particular, the diameter of the ultrasmall particles of iron oxide incorporated in the particles of the conjugate of the invention is comprised between 1 and 50 nm, more particularly between 1 and 30 nm.

[0023] For example, the diameter of the ultrasmall particles of iron oxide incorporated in the particles of the conjugate of the invention may be comprised between 1 and 45 nm, between 1 and 40 nm, between 1 and 35 nm, between 1 and 30 nm, between 2 and 50 nm, between 2 and 45 nm, between 2 and 40 nm, between 2 and 35 nm, between 2 and 30 nm, between 3 and 50 nm, between 3 and 45 nm, between 3 and 40 nm, between 3 and 35 nm, between 3 and 30 nm, between 5 and 50 nm, between 5 and 45 nm, between 5 and 40 nm, between 5 and 35 nm, between 5 and 30 nm, between 10 and 50 nm, between 10 and 45 nm, between

[0024] 10 and 40 nm, between 1 and 35 nm or between 10 and 30 nm.

[0025] Advantageously, the polymer matrix within which the utrasmall particles are embedded is biodegradable.

[0026] The polymer matrix within which the utrasmall particles are embedded is selected from polycathecolamines or polyserotonine. In particular, the biodegradable polymer matrix within which the utrasmall particles are embedded can be selected from polydopamine (PDA), polynorepinephrine (PNE), polyepinephrine (PEP) and polyserotonine, more particularly from polydopamine (PDA) and polynorepinephrine (PNE). Still more particularly, the biodegradable polymer matrix is poly dopamine (PDA).

[0027] In addition to their biodegradability, these different types of polymers have many advantages for the synthesis of the particles of the invention as well as their applications. In particular, a variety of ligand can be conjugated at high density with polycathecolamines or polyserotonine via Michael addition or Schiff base reactions (Lee, H. el al., Adv. Mater. 2009, 21, 431-434). The ability to conjugate a large amount of targeting moieties on the surface of the USPIO clusters is required to maximize binding to the target and reach a high sensitivity. Third, polycathecolamines or polyserotonine are hydrophilic and negatively charged at physiological pH, providing a negative zeta-potential for the coated particles and preventing their aggregation in solution. They form a strong bond to iron oxide which allows to obtain stable particles even under sonication, allowing dispersing aggregated particles without breaking the clusters or detaching the conjugated ligands.

[0028] In one embodiment, the iron oxide concentration in the particles of the suspension of particles comprised in the conjugate of the invention may be comprised between 50% and 95% in weight with respect to the total weight of the particles.

[0029] The iron concentration in the suspension of particles comprised in the conjugate of the invention may be determined by any suitable known by the person skilled in the art. In particular, the iron concentration in the particle of the invention may be determined by the ferrozine method or by mass spectrometry.

[0030] The particles of the suspension of particles comprised in the conjugate of the invention can be characterized by their polydispersity index. In one embodiment, the polydispersity index of the particles is below 0.3, in particular, the polydispersity index of the particles is comprised between 0.01 and 0.2. The polydispersity index of the particles may be determined by any suitable known by the person skilled in the art. In particular, the polydispersity index of the particles may be determined by dynamic light scattering (DLS) by using for example the same apparatus and measurements conditions as those used for the measurement of the hydrodynamic diameter.

[0031] For example, the poly dispersity index of the particles may be comprised between 0.01 and 0.3, between 0.02 and 0.3, between 0.03 and 0.3, between 0.05 and 0.3, between 0.07 and 0.3, between 0.1 and 0.3, between 0.01 and 0.2, between 0.02 and 0.2, between 0.03 and 0.2, between 0.05 and 0.2, between 0.07 and 0.2 or between 0.1 and 0.2.

[0032] The particles of the suspension of particles comprised in the conjugate of the invention can also be characterized by their zeta-potential. In one emdodiment, the zeta-potential of the particles is comprised between -50 and -20 mV, in particular between -45 and -25 mV, more particularly between -42 and -37 mV.

[0033] The zeta-potential may be determined by any suitable known by the person skilled in the art. In particular, the zeta-potential of the particle of the invention may be determined by electrophoretic light scattering (ELS) with a measurement carried out on the particles suspended in a 1 mM sodium chloride solution.

[0034] In the context of the invention, the term “zeta-potential” refers to the electrical potential at the interface which separates mobile fluid from fluid that remains attached to the surface of a particle.

[0035] The suspension of particles comprised in the conjugate according to the invention contains a solvent which can be selected from an aqueous solution, for example water, or a saline solution, or glycerol, or mannitol in which the particles of the invention described above are suspended.

[0036] In other words, the suspension of particles comprised in the conjugate according to the invention comprises particles suspended in a solvent selected from an aqueous solution, for example water, a saline solution, a glycerol solution and a mannitol solution. In particular, the suspension of particles comprises particles suspended in an aqueous solution or a saline solution. More particularly, the suspension of particles comprises particles suspended in an aqueous solution, preferably water, more preferably distilled water.

[0037] The mean hydrodynamic diameter of the particles in the suspension of particles comprised in the conjugate according to the invention is comprised between 250 and 900 nm.

[0038] Advantageously, the mean hydrodynamic diameter of the particles in the suspension of particles comprised in the conjugate according to the invention may be controlled.

[0039] In one embodiment, the mean hydrodynamic diameter of the particles in the suspension of particles is comprised between 250 and 400 nm, in particular between 250 and 350 nm, more particularly the mean hydrodynamic diameter of the particles in the suspension of particles is around 300 nm.

[0040] In another embodiment, the mean hydrodynamic diameter of the particles in the suspension of particles is comprised between 400 and 600 nm, in particular between 450 and 550 nm, more particularly the mean hydrodynamic diameter of the particles in the suspension of particles is around 500 nm.

[0041] In another embodiment, the mean hydrodynamic diameter of the particles in the suspension of particles is comprised between 600 and 900 nm, in particular between 650 and 750 nm, more particularly the mean hydrodynamic diameter of the particles in the suspension of particles is around 700 nm.

[0042] The suspension of particles comprised in the conjugate according to the invention may be prepared by a process comprising the steps of: a) preparing a suspension of ultrasmall particles of iron oxide, in particular having a diameter between 1 and 50 nm, more particularly between 1 and 30 nm; b) coating of the ultrasmall particles of iron oxide with a catecholamine or serotonine, in particular with a catecholamine, more particularly with dopamine; c) polymerizing the catecholamine or serotonine in the presence of ultrasmall particles of iron oxide; d) terminating said polymerization; e) recovering a suspension of particles.

[0043] The step a) of the process consists in preparing a suspension of ultrasmall particles of iron oxide, in particular in water, more particularly in distilled water. In particular, the ultrasmall particles of iron oxide may be chosen among maghemite of formula FC2O3, magnetite of formula FC3O4 or a mixture of FC2O3 and FcaCF. More particularly, the ultrasmall particles of iron oxide are FcaCF.

[0044] The step a) of the process may be performed according to any suitable method known by the person skilled in the art, including, for example, co-precipitation, solvothermal synthesis, thermal decomposition, polyol process, sonochemical reaction and sol-gel reaction. In particular, the step a) of the process may by performed by a co-precipitation method in an alkaline buffer. Typically, the co-precipitation method is perfomed by mixing FeCh and FeCh, preferably in a molar ratio of 2:1, in an alkaline buffer, and adding progressively an ammonia solution, said ammonia solution being preferably at a concentration ranging from 10% to 15% (w / v), more preferably at a concentration of 13% (w / v), in particular at a rate ranging from 0.1 to 0.3 mL / min, more particularly from 0.15 to 0.25 mL / min, still more particularly at a rate of 0.2 mL / min, until precipitation of the iron oxide. The precipitate is optionally washed, preferably with distilled water, and resuspended, preferably in distilled water.

[0045] The step b) of the process consists in coating the ultrasmall particles of iron oxide with a catecholamine or serotonine. Typically, the suspension of ultrasmall particles of iron oxide obtained from step a) is mixed with a solution of catecholamine or serotonine, in particular a solution of catecholamine, more particularly a solution of dopamine, preferably in a weight ratio iron / catecholamine or serotonine comprised between 0.5 and 3.0, in particular between 0.5 and 2.5, more particularly between 1.0 and 2.0, still more particularly between 1.0 and 1.5, even more particularly a mass ratio iron / catecholamine or serotonine of 1.2.

[0046] After mixing the suspension of ultrasmall particles of iron oxide obtained from step a) with a solution of catecholamine or serotonine, the step b) of the process of the invention may include a step of homogeneization of the resulting mixture, in particular by sonication, for example using a UP200ST sonicator (Hielscher) at 70% amplitude and 26 KHz, particularly during 1 to 30 minutes, more particularly 10 to 20 minutes, at a temperature ranging from 0°C to 100°C, preferably 40°C to 100°C.

[0047] The step b) of the process may further include a step of centrifugation, in particular at 3000G.

[0048] The step c) of the process consists in polymerizing the catecholamine or serotonine, in particular a catecholamine, more particularly dopamine, in which the ultrasmall particles of iron oxide are coated.

[0049] The polymerization step may be performed by any suitable method known the skilled person in the art. In particular, the polymerization step may be performed by adding a base solution, preferably ammonia, and oxygen, preferably oxygen from the ambient air, to the ultrasmall particles of iron oxide coated with a catecholamine or serotonine obtained in step b) and stirring the resulting mixture for 15 minutes to 6 hours, preferably 30 minutes to 90 minutes, more preferably for 60 minutes. This leads to the self-assembly the ultrasmall particles of iron oxide with the polycatecholamine or polyserotonine.

[0050] The amount of ammonia added during step c) allows to control the size of the particles of the suspension of particles.

[0051] In one embodiment, the ammonia added during step c) is comprised between 2.17 and 3.25 parts in weight with respect with 1 part of iron. Typically, between 2.0 mL and 3.0 mL of a 13% (w / v) ammonia solution is added for 120 mg of iron.

[0052] According to this embodiment, the mean hydrodynamic diameter of the particles is comprised between 250 and 400 nm, in particular between 250 and 350 nm, more particularly the mean hydrodynamic diameter of the particles is around 300 nm.

[0053] In another embodiment, the amount of ammonia added during step c) is comprised between 0.33 and 2.17 parts in weight with respect to 1 part of iron. Typically, between 0.3 mL and 2.0 mL of a 13% (w / v) ammonia solution is added for 120 mg of iron. According to this embodiment, the mean hydrodynamic diameter of the particles is comprised between 400 and 600 nm, in particular between 450 and 550 nm, more particularly the mean hydrodynamic diameter of the particles is around 500 nm.

[0054] In another embodiment, the amount of ammonia added during step c) is comprised between 0.05 and 0.33 parts in weight with respect to 1 part of iron. Typically, between 0.05 mL and 0.3 mL of a 13% (w / v) ammonia solution is added for 120 mg of iron.

[0055] According to this embodiment, the mean hydrodynamic diameter of the particles is comprised between 600 and 750 nm, in particular between 650 and 750 nm, more particularly the mean hydrodynamic diameter of the particles is around 700 nm.

[0056] The step d) of the process consists in terminating the polymerization of step c).

[0057] The termination step may be performed by any suitable method known to the skilled person in the art, such as, for example, the addition of an acid to get an acidic pH in the reaction mixture, the addition of a polymerization inhibitor, or replacing the reaction medium. In particular, the termination step is performed by replacing the reaction medium. Typically, the particles are separated from the solution of base in which the polymerization step c) was performed, in particular using a separating magnet or a centrifugation step, more particularly a centrifugation step, allowing to keep the particles in a bottom layer and the base solution as a supernatant. The supernatant is then removed and replaced by a wash solution, in particular water, more particularly distilled water. This operation can be done multiple times to ensure that the base solution from step c) is totally removed and replaced by the wash solution.

[0058] The step e) of the process consists in the recovery of a suspension of the particles of the invention. These particles can then be stored at a low temperature of about 3 to 10°C, either in water, or saline solution.

[0059] Prior to its injection to a patient, the suspension of particles is kept under agitation at a temperature of 0 to 37 °C, in particular 4°C. In one embodiment, particles may be obtained by the above process for preparing a suspension of particles followed by an additional step of isolation of said particles by removal of the solvent of the suspension obtained after step e).

[0060] The conjugate of the invention comprises a suspension of particles as defined above and a protein that can be an activator of fibrinolysis or a fragment thereof.

[0061] Typically, the activator of fibrinolysis comprises free amine or a thiol groups and is linked to the particles of the suspension of particles via said free amine or a thiol groups.

[0062] In an embodiment, the activator of fibrinolysis may be modified with a linker comprising at least a free amine or a thiol group. In other words, the activator of fibrinolysis may be linked to the particles of the suspension of particles through a linker moiety comprising at least a free amine or a thiol group.

[0063] In the context of the invention, the term “linker” or “linker moiety” refers to a connector for linking a molecule to particles of the suspension of particles.

[0064] Particularly, the activator of fibrinolysis may be a tissue plasminogen activator (tPA). More particularly, the activator of fibrinolysis may be a recombinant tissue plasminogen activator (rtPA). Still more particularly, the recombinant tissue plasminogen activator may be alteplase, reteplase, urokinase, tenecteplase, desmatoplase, streptokinase or optimized tPA (OptPA). Even more particularly, the recombinant tissue plasminogen activator may be alteplase, urokinase or tenecteplase. In a particular example, the recombinant tissue plasminogen activator may be alteplase.

[0065] In one embodiment, the activator of fibrinolysis may be selected from alteplase, reteplase, urokinase, tenecteplase, desmatoplase, streptokinase and optimized tPA (OptPA). In particular, the activator of fibrinolysis may be selected from alteplase, reteplase, urokinase, tenecteplase, desmatoplase and streptokinase. More particularly, the activator of fibrinolysis may be selected from alteplase, reteplase, urokinase and Tenecteplase. Still more particularly, the activator of fibrinolysis may be selected from alteplase, urokinase and tenecteplase. Even more particularly, the activator of fibrinolysis may be alteplase. The amount of particles in the conjugate of the invention may be comprised between 10% and 35%, in particular between 20% and 35%, more particularly between 25% and 35%, still more particularly between 30% and 35%, in weight with respect to the total weight of the conjugate.

[0066] In a particular embodiment, the conjugate of the invention may further comprise a deoxyribonuclease (DNAse).

[0067] The amount of deoxyribonuclease in the conjugate of the invention may be comprised between 15% and 40%, in particular between 15% and 35%, more particularly between 20% and 35%, still more particularly between 20% and 30%, in weight with respect to the total weight of the conjugate.

[0068] The conjugate of the invention may be used as a suspension in a physiological medium compatible with an injection to a human patient.

[0069] The present invention thus also relates to a suspension comprising the conjugate of the invention and a physiological medium, in particular a physiological medium compatible with an injection to a human patient.

[0070] In particular, the physiological medium may be mannitol or glycerol, more particularly a mannitol solution or a glycerol solution, even more particularly a 0.3 M mannitol solution.

[0071] Advantageously, the concentration of tissue plasminogen activator in the suspension comprising the conjugate of the invention and a physiological medium is comprised between 0.3 mg / mL and 0.6 mg / mL, in particular between 0.3 mg / mL and 0.5 mg / mL, more particularly between 0.4 mg / mL and 0.6 mg / mL, even more particularly between 0.4 mg / mL and 0.5 mg / mL.

[0072] The suspension comprising the conjugate of the invention and a physiological medium may be prepared according to the following process: a) Providing a solution of tissue plasminogen activator; b) Providing a suspension of particles as defined above; c) Mixing the solution of tissue plasminogen activator and the suspension of particles; d) Incubating the mixture obtained in step c) at temperature comprised between 2°C and 20°C, in particular between 2°C and 15°C, more particularly between 2°C and 10°C, still more particularly between 3°C and 7°C, even more particularly between 3°C and 5°C, preferably at 4°C; e) Recovering the conjugate obtained in step d); f) Resuspending the conjugate in a physiological medium, in particular a physiological medium compatible with an injection to a human patient.

[0073] Optionally, the tissue plasminogen activator is dialyzed before performing step a) of the above process.

[0074] Advantageously, the tissue plasminogen activator concentration in the solution of tissue plasminogen activator of step a) is comprised between 1.5 and 2.0 mg / mL, in particular between 1.5 and 1.9 mg / mL, more particularly between 1.5 and 1.8 mg / mL, still more particularly between 1.6 and 1.7 mg / mL.

[0075] Advantageously, the iron concentration in the suspension of particles of step b) is comprised between 0.5 and 1.0 mg / mL, in particular between 0.6 and 1.0 mg / mL, more particularly between 0.7 and 1.0 mg / mL, still more particularly between 0.8 and 0.9 mg / mL.

[0076] Advantageously, the tissue plasminogen activator concentration in the mixture obtained in step c) is comprised between 0.5 and 1.0 mg / mL, in particular between 0.6 and 1.0 mg / mL, more particularly between 0.7 and 1.0 mg / mL, still more particularly between 0.8 and 0.9 mg / mL.

[0077] Advantageously, the iron concentration in the mixture obtained in step c) is comprised between 0.25 and 0.5 mg / mL, in particular between 0.3 and 0.5 mg / mL, more particularly between 0.35 and 0.5 mg / mL, still more particularly between 0.4 and 0.45 mg / mL.

[0078] Advantageously, step d) of the above process is performed for a period of time comprised between 45 min and 2 h, in particular between 45 min and 1 h 30 min, more particularly between 50 min and 1 h 20 min, still more particularly between 50 min and 1 h 10 min. In a particular example, step d) of the above process is performed for 1 h. Prior to their injection, the conjugate of the invention can be resuspended in any physiological medium compatible with an injection to a human patient, according to step f) of the above process. The physiological medium of step f) of the above process may be mannitol or glycerol, in particular a mannitol solution or a glycerol solution, more particularly a 0.3 M mannitol solution.

[0079] Advantageously, the final concentration of tissue plasminogen activator in the suspension of conjugate obtained in step f) is comprised between 0.3 mg / mL and 0.6 mg / mL, in particular between 0.3 mg / mL and 0.5 mg / mL, more particularly between 0.4 mg / mL and 0.6 mg / mL, even more particularly between 0.4 mg / mL and 0.5 mg / mL.

[0080] After their preparation, the conjugates of the invention are kept under agitation at a temperature of 0 to 37 °C, in particular 4°C, until their injection to a patient.

[0081] When the conjugate of the invention further comprises a deoxyribonuclease, the suspension comprising the conjugate of the invention and a physiological medium may be prepared according to the following process: a) Providing a solution of tissue plasminogen activator; b) Providing a suspension of particles as defined above; c) Mixing the solution of tissue plasminogen activator and the suspension of particles; d) Incubating the mixture obtained in step c) at temperature comprised between 2°C and 20°C, in particular between 2°C and 15°C, more particularly between 2°C and 10°C, still more particularly between 3°C and 7°C, even more particularly between 3°C and 5°C, preferably at 4°C; e) Recovering the conjugate obtained in step d); f) Mixing the conjugate obtained in step e) with a solution of deoxyribonuclease; g) Incubating the mixture obtained in step f) at temperature comprised between 2°C and 20°C, in particular between 2°C and 15 °C, more particularly between 2°C and 10°C, still more particularly between 3°C and 7°C, even more particularly between 3°C and 5°C, preferably at 4°C; h) Recovering the conjugate obtained in step g); i) Resuspending the conjugate in a physiological medium, in particular a physiological medium compatible with an injection to a human patient.

[0082] In order to be used in the detection and the treatment of microthrombi during acute ischemic stroke in magnetic resonance imaging, the conjugate as defined above has to be administered to a patient prior to the imaging step. The conjugate as defined above may be administered as a formulation in an effective amount by any of the accepted modes of administration, preferably by intravenous, intraarterial or oral route, more preferably by intravenous route.

[0083] The conjugate as defined above or the suspension comprising the conjugate as defined above is useful for detecting microthrombi during acute ischemic stroke in magnetic resonance imaging because of the iron oxide composing the particles that generate a distinct hypotense signal on T2*-weigthed magnetic resonance imaging in the presence of microthrombi. The poly dopamine matrix also confer targeting properties to thrombosis.

[0084] The present invention thus relates to the conjugate as defined above, for use in an in vivo method of detection of microthrombi in a condition requiring thrombolysis, comprising the steps of: a) administering to a patient the conjugate as defined above or the suspension comprising the conjugate as defined above; b) detecting the microthrombi by magnetic resonance imaging (MRI).

[0085] In one embodiment, the condition requiring thrombolysis is selected from acute ischemic stroke, myocardial infarction, pulmonary embolism, deep vein thrombosis, renal vein thrombosis, carotid thrombosis, portal vein thrombosis, mesenteric vein thrombosis, and local fibrinolytic therapy in intracranial hemorrhages. In particular, the condition requiring thrombolysis is acute ischemic stroke.

[0086] In other terms, the present invention also relates to an in vivo method of detection of microthrombi in a condition requiring thrombolysis, comprising the steps of: a) administering an effective amount of the conjugate as defined above, to a patient in need thereof; b) detecting the microthrombi by magnetic resonance imaging (MRI).

[0087] Preferably the patient is a warm-blooded animal, more preferably a human.

[0088] In one embodiment, when the patient is a human, the conjugate of the invention is administered to the patient in an amount comprised between 0.1 mg / kg and 1.0 mg / kg, with respect to to the weight of the patient. For example, the conjugate of the invention may be administered to the patient in an amount comprised between 0.1 mg / kg and 0.9 mg / kg, between 0.1 mg / kg and 0.8 mg / kg, between 0.1 mg / kg and 0.7 mg / kg, between 0.1 mg / kg and 0.6 mg / kg, between 0.1 mg / kg and 0.5 mg / kg, between 0.1 mg / kg and 0.4 mg / kg, between 0.1 mg / kg and 0.3 mg / kg, between 0.1 mg / kg and 0.2 mg / kg, between 0.2 mg / kg and 1.0 mg / kg, between 0.3 mg / kg and 1.0 mg / kg, between 0.4 mg / kg and 1.0 mg / kg, between 0.5 mg / kg and 1.0 mg / kg, between 0.6 mg / kg and 1.0 mg / kg, between 0.7 mg / kg and 1.0 mg / kg, between 0.8 mg / kg and 1.0 mg / kg, or between 0.9 mg / kg and 1.0 mg / kg, with respect to the weight of the patient.

[0089] For information, in a mouse, the conjugate of the invention is administered to the patient in an amount comprised between 1.0 mg / kg and 10.0 mg / kg, with respect to to the weight of the mice.

[0090] The detection of the microthrombi by magnetic resonance imaging is performed by brain MRI of the patient with T2*-weighted sequences. In particular, the sequences are 3D T2*- weighted gradient echo imaging with flow compensation (GEFC, spatial resolution of 93 x 70 x 70 pm interpolated to an isotropic resolution of 70 pm) with TE / TR 9 / 50 ms and a flip angle of 15°. The conjugate generates a distinct hypointense signal corresponding to the micro thrombi.

[0091] The conjugate as defined above is useful for treating microthrombi during acute ischemic stroke because the functionalization with tPA enhance fibrinolysis activity. The specific targeting of the microthrombi by the conjugate enhance the local activity of tPA at the edge of the microthrombi. The microthrombi lysis by the tPA present on the particles can be monitored during magnetic resonance imaging acquisitions. The poly dopamine confers antioxidant properties that protect neurons from damages induced by deprivation of glucosis and oxygen.

[0092] In addition, the use of the conjugate of the invention resulted in a decreased brain lesion size in a mouse ischemic stroke model, in particular after 24 hours of treatment. In other words, the conjugate of the invention allows a decrease of the brain lesion size in a mouse ischemic stroke model, in particular after 24 hours of treatment.

[0093] In particular, when the conjugate of the invention further comprises a deoxyribonuclease, the use of the conjugate of the invention resulted in a homogeneous reduction in lesion size in a mouse ischemic stroke model, in particular after 24 hours of treatment.

[0094] The invention thus also relates to the conjugate of the invention or the suspension of the invention, for use in the treatment of a condition requiring thrombolysis.

[0095] In one embodiment, the invention relates to the conjugate of the invention, for use in the treatment of a condition requiring thrombolysis.

[0096] In one embodiment, the invention relates to the suspension of the invention, for use in the treatment of a condition requiring thrombolysis.

[0097] In one embodiment, the condition requiring thrombolysis is selected from acute ischemic stroke, myocardial infarction, pulmonary embolism, deep vein thrombosis, renal vein thrombosis, carotid thrombosis, portal vein thrombosis, mesenteric vein thrombosis, and local fibrinolytic therapy in intracranial hemorrhages. In particular, the condition requiring thrombolysis is acute ischemic stroke.

[0098] In one embodiment, said treatment of a condition requiring thrombolysis is monitored by magnetic resonance imaging (MRI).

[0099] In other terms, the present invention also relates to a method of treating a condition requiring thrombolysis comprising the administration of a therapeutically effective amount of a conjugate as defined above or a suspension as defined above, to a patient in need thereof. Preferably the patient is a warm-blooded animal, more preferably a human. The invention further provides the use of a conjugate or a suspension as defined above for the manufacture of a medicament for use in treating a condition requiring thrombolysis.

[0100] The invention thus also relates to the conjugate of the invention or the suspension of the invention, for use in the treatment of microthrombi during acute ischemic stroke.

[0101] In one embodiment, the invention relates to the conjugate of the invention, for use in the treatment of microthrombi during acute ischemic stroke.

[0102] In one embodiment, the invention relates to the suspension of the invention, for use in the treatment of microthrombi during acute ischemic stroke.

[0103] In other terms, the present invention also relates to a method of treating microthrombi during acute ischemic stroke, comprising the administration of a therapeutically effective amount of a conjugate as defined above or a suspension as defined above, to a patient in need thereof. Preferably the patient is a warm-blooded animal, more preferably a human.

[0104] The invention further provides the use of a conjugate or a suspension as defined above for the manufacture of a medicament for use in treating microthrombi during acute ischemic stroke.

[0105] In one embodiment, said treatment of microthrombi is monitored by magnetic resonance imaging (MRI).

[0106] The present invention is also directed to an in vivo diagnostic method using the conjugate or the suspension as defined above.

[0107] The present invention also relates to the conjugate as defined above or the suspension as defined above for use in the treatment of acute ischemic stroke.

[0108] In one embodiment, the present invention relates to the conjugate as defined above, for use in the treatment of acute ischemic stroke.

[0109] In one embodiment, the present invention relates to the suspension as defined above, for use in the treatment of acute ischemic stroke. In other terms, the present invention also relates to a method of treating acute ischemic stroke, comprising the administration of a therapeutically effective amount of a conjugate as defined above or a suspension as defined above, to a patient in need thereof. Preferably the patient is a warm-blooded animal, more preferably a human.

[0110] The invention further provides the use of a conjugate as defined above or a suspension as defined above for the manufacture of a medicament for use in treating acute ischemic stroke.

[0111] DEFINITIONS

[0112] The definitions and explanations below are for the terms as used throughout the entire application, including both the specification and the claims.

[0113] When describing the particles of the invention, the terms used are to be construed in accordance with the following definitions, unless indicated otherwise.

[0114] The term “biocompatible”, as used herein, refers to materials that do not cause significant harm to living tissue when placed in contact with such tissue, e.g., in vivo. In certain embodiments, materials are “biocompatible” if they are not toxic to cells. In certain embodiments, materials are “biocompatible” if their addition to cells in vitro results in less than or equal to 20% cell death, and / or their administration in vivo does not induce significant inflammation or other such adverse effects.

[0115] The term “biodegradable” as used herein refers to materials that, when introduced into cells, are broken down (e.g., by cellular machinery, such as by enzymatic degradation, by hydrolysis, and / or by combinations thereof) into components that cells can either reuse or dispose of without significant toxic effects on the cells. In certain embodiments, components generated by breakdown of a biodegradable material are biocompatible and therefore do not induce significant inflammation and / or other adverse effects in vivo. In some embodiments, biodegradable polymer materials break down into their component monomers. In some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymer materials) involves hydrolysis of ester bonds. Alternatively or additionally, in some embodiments, breakdown of biodegradable materials (including, for example, biodegradable polymer materials) involves cleavage of urethane linkages. Exemplary biodegradable polymers in the context of the present invention include, for example, polydopamine (PDA), polynorepinephrine (PNE), polyepinephrine (PEP) and polyserotonine (PST), polydopamine (PDA) being particularly preferred.

[0116] As used herein, the term “embedded” relative to nanoparticles and the surface of the polymer matrix refers to the nanaoparticles being at least partially extended into the surface such that the polymer is in contact with the nanoparticle to a greater degree than would accur if the nanoparticles were simply laid on the surface of the polymer matrix.

[0117] The term “suspension” as used herein refers to a heterogeneous mixture of materials comprising a liquid and a finely dispersed solid material.

[0118] The term “patient” refers to a warm-blooded animal, more preferably a human, who / which is awaiting or receiving medical care or is or will be the object of a medical procedure.

[0119] The term “human” refers to subjects of both genders and at any stage of development (i.e. neonate, infant, juvenile, adolescent, adult). In one embodiment, the human is an adolescent or adult, preferably an adult.

[0120] The term “administration”, or a variant thereof (e.g., “administering”), means providing the active agent or active ingredient, alone or as part of a pharmaceutically acceptable composition, to the patient in whom / which the condition, symptom, or disease is to be treated.

[0121] By “pharmaceutically acceptable” is meant that the ingredients of a pharmaceutical composition are compatible with each other and not deleterious to the patient thereof.

[0122] The term “excipient” as used herein means a substance formulated alongside the active agent or active ingredient in a pharmaceutical composition or medicament. Acceptable excipients for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, 21stEdition 2011. The choice of excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense of being not deleterious to the recipient thereof. The at least one pharmaceutically acceptable excipient may be for example, a binder, a diluent, a carrier, a lubricant, a disintegrator, a wetting agent, a dispersing agent, a suspending agent, and the like.

[0123] The term “pharmaceutical vehicle” as used herein means a carrier or inert medium used as solvent or diluent in which the pharmaceutically active agent is formulated and / or administered. Non-limiting examples of pharmaceutical vehicles include creams, gels, lotions, solutions, and liposomes.

[0124] The present invention will be better understood with reference to the following examples and figures. These examples are intended to be representative of specific embodiments of the invention, and are not intended as limiting the scope of the invention.

[0125] FIGURES

[0126] Figure 1. IO@PDA@tPA characterization and amidolytic capacities test in vitro. A. Grafting tPA to the surface of poly dopamine iron oxide particles (IO@PDA@tPA) protocol design. B. Summary table of tPA loaded on the IO @ PDA, the IO @ PDA size and charge (n=49). C. Representative spectrofluor test curves showing how tPA activity is deduced. D. Spectrofluor test demonstrates that tPA amidolytic activity remains the same when link to IO@PDA (2-way ANOVA, multiple comparisons, n=5 per group).

[0127] Figure 2. IO@PDA@tPA thrombolytics capacities test in vitro. A. Representative clot lysis assay curves showing how 75% clotting time (75% CT) and 50% lysis time (50% LT) were measured. B. Mean 75% CT of human plasma in the presence of different concentrations of IO@PDA@tPA shows no differences between the groups (2- way ANOVA, multiple comparisons, n=5 per group). C. Mean 50% LT of human plasma in the presence of different concentrations of IO@PDA@tPA shows no differences between the groups (2-way ANOVA, multiple comparisons, n=5 per group). D. Representative Halo assay curves showing how activation time (At); maximum rate of clot lysis (CLRmax) and 50% clot lysis were measured. E. Graphical representations of the activation time, CLRmax and 50% lysis show no differences between the groups (Kruskall- Wallis test, multiple comparison, n=5 per group). Figure 3. IO@PDA@tPA theranostic application in vivo. A. Test groups. B. Microthrombi quantification after injection in a mouse model of ischemic stroke. IO@PDA@tPA hyposignal significantly decreased compared to IO @ PDA (2- way ANOVA, multiple comparisons (n=3-4). C. Re-injection of IO@PDA alone after IO@PDA@tPA treatment in MRI confirms that the loss of signal by MRI is due to IO@PDA@tPA and not because of a non-recognition of the microthrombi by the particles.

[0128] Figure 4. IO@PDA@tPA improves outcome at 24h post ischemic event. A. Test groups and in vivo protocol design. B. Lesion size at 24 h after injection of IO @ PDA; IO@PDA@tPA 2.5 mg / kg; IO@PDA@tPA 5 mg / kg; IO@PDA + tPA 2.5 mg / kg or tPA (lO mg / kg) infusion. IO@PDA@tPA 2.5 mg / kg significantly decrease the lesion volume compared to other groups and show no differences with the tPA (10 mg / kg) infusion treated mice (One-way ANOVA, multiple comparisons, n=4-12). C. Angiographies show a majority of no recanalization of the MCA in IO@PDA and IO@PDA + tPA 2.5 mg / kg conditions but a majority of complete recanalization at 24 h after IO@PDA@tPA 2.5 mg / kg and 5 mg / kg administration. D. Functional recovery: evaluation of the left paw strength compared to the right paw. Mice treated with IO@PDA@tPA present a full recovery at 5 days after stroke whereas the IO @ PDA and IO @ PDA + tPA 2.5 mg / kg treated groups still have functional deficits (2- way ANOVA, multiple comparisons, n=5-10).

[0129] Figure 5. Other plasminogen activators can be linked to the IO @ PDA and conserve their properties. A. Summary table of the iron concentrations of the IO @ PDA, the concentration of plasminogen activators added to the IO @ PDA solution and the concentration of plasminogen activators, in the supernatant and fixed on the IO @ PDA. B. Representative curves of tPA (1; 2.5 and 5 nM) and IO@PDA@tPA (1; 2.5 and 5 nM). C. Representative curves of urokinase (1; 2.5 and 5 nM) and IO @ PDA @ urokinase (1; 2.5 and 5 nM). D. Representative curves of TNK (1; 2.5 and 5 nM) and IO@PDA@TNK (1; 2.5 and 5 nM).

[0130] Figure 6. Synthesis and characterization of the IO@PDA@tPA. A. Schematic illustration of the IO@PDA@tPA synthesis. Briefly, IO @ PDA were first synthetized, with a co-precipitation of Fe2+ / Fe3+that led to nanocrystal formation, the dopamine in alkaline condition polymerized into polydopamine facilitating the clustering of Fe2+ / Fe3+into the matrix. Then, dialyzed tPA was mixed with the 10 @ PDA leading to the formation of the IO@PDA@tPA. B. DLS analysis of 10 @ PDA and IO@PDA@tPA, provided the mean hydrodynamic diameter (n=3 particles preparations). C. Transmission electron microscopy images of I0@PDA and IO@PDA@tPA (scale bar = 0.22 pm). D. I0@PDA were functionalized with tPA grafted with an Alexa Fluor 488. Confocal microscopy confirmed the functionalization of the tPA (green) on the IO @ PDA (darkfield).

[0131] Figure 7. Optimization of the tPA Coupling Method on Particles. A-B. Final IO@PDA@tPA optimization with dialyzed tPA with IO@PDA@tPA resuspension in Mannitol (0.3M) demonstrated no loss of tPA activity (A) and a strong correlation between the amount of tPA and its activity. There was no difference between the two-correlation slope reinforcing the precedent results (B). C-D. First studies using non- dialyzed tPA showed a loss of tPA activity when grafted to the 10 @ PDA particles (C). The correlation slope strongly supported a loss of amyloidic activity of non-dialyzed tPA when conjugated on the 10 @ PDA (D). E-F. Other attempts with dialyzed tPA with IO@PDA@tPA resuspension in HEPES (0.3M) instead of mannitol (0.3M) again revealed a loss of tPA activity when coupled to the 10 @ PDA (E, F). G-H. Finally, increasing the concentration of I0@PDA 2-fold higher than the concentration of tPA showed once again a loss of tPA activity when linked on the particles (G, H).

[0132] Figure 8. Theranostic treatment of microthrombi in comorbidity diabetic mice. A. Protocol illustration of the intravenous injection of I0@PDA; IO@PDA@tPA 2.5 mg / kg; I0@PDA + tPA 2.5 mg / kg; tPA 10 mg / kg; I0@PDA + tPA 10 mg / kg 20 minutes after the thromboembolic stroke model. T2- weighted images for the lesion size, T2s-weighted images for hemorrhagic transformation and TOF sequences for the vasculature were acquired 24 hours and 5 days after stroke onset by MRI. Finally, to assess functional recovery, grip test was performed the day before inducing the ischemic stroke and at day 1 and day 5 after stroke. B. Lesion size 24h after injection of I0@PDA; IO@PDA@tPA 2.5 mg / kg; I0@PDA + tPA 2.5 mg / kg; tPA 10 mg / kg; I0@PDA + tPA 10 mg / kg. IO@PDA@tPA 2.5 mg / kg significantly decreased the lesion volume compared to the IO @ PDA group and had a tendency of decreasing the lesion size compared to the other groups (One-way ANOVA, multiple comparisons, n=9-12). C. Angiographies showed that one third of the mice had complete recanalization for IO@PDA@tPA 2.5 mg / kg at 24h after stroke whereas in the IO @ PDA group, a majority of animals showed no recanalization of the MCA 24h after stroke. The other groups showed between 60 and 81.8% of partial recanalization and no recanalization at 24h after stroke. D. Angiographies 5 days after stroke revealed a majority of complete recanalization for mice treated with IO@PDA@tPA 2.5 mg / kg. In the other groups, the majority of animals showed partial recanalization of the MCA or no recanalization at all in the IO@PDA; IO@PDA + tPA 2.5 mg / kg; and IO@PDA + tPA 10 mg / kg groups. E. Representation of the hemorrhagic transformation scale for the analysis. F. T2s-weighted acquisitions revealed no hemorrhagic transformation at 5 days after stroke for the IO@PDA@tPA 2.5 mg / kg whereas all the other groups showed some level of hemorrhagic transformation (between 9.1% to 22.2%). G. Quantification of the global strength deficit measured by a grip-test of the forepaws of the mice treated with IO @ PDA; IO@PDA@tPA 2.5 mg / kg; IO@PDA + tPA 2.5 mg / kg; tPA 10 mg / kg; IO@PDA + tPA 10 mg / kg before IS and at 1 and 5 days after IS stroke (2way ANOVA, multiple comparisons, n=9-12 per group). H. Quantification of the specific left paw strength deficit measured by a grip-test ratio (strength of the left paw relative to the right paw) of the mice treated with IO@PDA; IO@PDA@tPA 2.5 mg / kg; IO@PDA + tPA 2.5 mg / kg; tPA 10 mg / kg; IO@PDA + tPA 10 mg / kg before IS and at 1 and 5 days after IS stroke (2way ANOVA, multiple comparisons, n=9-12 per group).

[0133] Figure 9. No hemorraghic transformation at 24h post IS. T2s acquisitions revealed no hemorrhagic transformation at 24h after stroke under any conditions. Nevertheless, a hypointense signal corresponding to the presence of microthrombi in the IO @ PDA and IO@PDA + tPA 2.5 mg / kg was observed at 24 hours after stroke. This highlighted that tPA at 2.5 mg / kg needed to be grafted on IO @ PDA to be more efficient.

[0134] Figure 10. Mice treated with IO@PDA@tPA showed better sensorimotor recovery in diabetic mice. A. Representation of the corridor device. B. Quantification of the visited objects on the left side by the mice treated with IO@PDA; IO@PDA@tPA 2.5 mg / kg; IO @ PDA + tPA 2.5 mg / kg; tPA 10 mg / kg; IO @ PDA + tPA 10 mg / kg before IS and at 1 and 5 days after IS (2way ANOVA, multiple comparisons, n=9-12 per group). C-E. More detailled representation of the visited objects on the left side before IS (C), at 1 day (D) and 5 days after IS (E) (contingency table, n=9-12).

[0135] Figure 11. IO @ PDA @tPA@ DNase synthesis and characterization. A. Schematic illustration of the IO @ PDA @tPA@ DNase synthesis. Briefly, IO @ PDA were first synthesized as described before. Then, dialyzed tPA was mixed with the IO @ PDA leading to the formation of the IO@PDA@tPA, then the DNase- 1 was added leading to the new conjugate synthesis: the IO @ PDA @tPA@ DNase. B. Table summarizing the concentration of tPA and DNase present at the surface of the IO@PDA, n=10. C. Representative DNA electrophoresis demonstrating that the DNAse keeps its enzymatic properties intact even grafted on the IO@PDA@tPA. D-F. Representative curves (D) and slopes (E) obtained after the spectrofluorometric assay. Quantification showed that tPA amidolytic properties remain intact event with the presence of DNase (F).

[0136] Figure 12. IO @ PDA @tPA@ DNAse are more efficient at lysing patient-derived thrombi than tPA or IO@PDA@tPA. A. Schematic representation of the experimental design. B-D. Clot weight loss over time for individual paired comparisons: free tPA vs IO@PDA@tPA (B), free tPA + DNase vs IO @ PDA @tPA@ DNase (C), IO@PDA@tPA vs IO @ PDA @tPA@ DNase (D). E-G; Quantification of final clot lysis percentage for each comparison. Statistical analysis was performed using one-way ANOVA with pairwise comparisons (n = 5).

[0137] Figure 13. IO @ PDA @tPA@ DNAse promotes early reperfusion. A. Schematic representation of the experimental design. Briefly, we used functional Ultrasound (fUS) imaging to quantify reperfusion in the ischemic area. Before inducing the IS, a 5-min acquisition was performed to have the baseline (TO) cerebral blood volume (CBV) of the mice. Then IS was induced by applying A1C13on the MCA for 5 minutes. 15 minutes after, a 45-minute acquisition began to follow any changes in the CBV in the ischemic area. 5 minutes after the beginning of the 45-minutes acquisition, one of the treatments: IO@PDA (2mg / kg); DNase (1.37 mg / kg); tPA (10 mg / kg); a combination of IO@PDA (2 mg / kg) + tPA (2.5 mg / kg) + DNase (1.37 mg / kg) and the IO @ PDA @tPA@ DNase conjugate (corresponding to 2.5 mg / kg tPA and 1.37 mg / kg DNase equivalency) was administered to the mice (corresponding to 20 minutes post ischemic events). B. CBV variation of the ipsilateral isocortex of IO @ PDA-treated mice (percentage from baseline (TO), n=5, 2-way ANOVA, Multiple comparisons (TO vs Tn+1), **** p < 0.0001). C. CBV variation of the ipsilateral isocortex DNase-treated mice (percentage from baseline (TO), n=6, 2-way ANOVA, Multiple comparisons (TO vs Tn+1), **** p < 0.0001). D. CBV variation of the ipsilateral isocortex tPA-treated mice (percentage from baseline (TO), n=5, 2-way ANOVA, Multiple comparisons (TO vs Tn+1), **** p < 0.0001). E. CBV variation of the ipsilateral isocortex IO@PDA + tPA + DNase-treated mice (percentage from baseline (TO), n=5, 2- way ANOVA, Multiple comparisons (TO vs Tn+1), **** p < 0.0001). F. CBV variation of the ipsilateral isocortex IO @ PDA @tPA@ DNase-treated mice (percentage from baseline (TO), n=5, 2-way ANOVA, Multiple comparisons (TO vs Tn+1), **** p < 0.0001). G. Representative images obtained during baseline (TO); 20 minutes and 60 minutes post IS for IO @ PDA @tPA@ DNase-treated mice.

[0138] Figure 14. IO @ PDA @tPA@ DNase decreases the lesion size and promotes complete recanalization at 24 hours post ischemic event. A. Lesion volume (mm3) at 24 h after the injection of saline, IO@PDA, DNase, tPA (10 mg / kg), IO@PDA + tPA (2.5 mg / kg) + DNase or IO @ PDA @tPA@ DNase. B. Illustrations of the lesion site area obtained after T2- weighted MRI. C. Angiography reveals a majority of non-recanalization of the MCA at 24h for mice treated with IO@PDA, DNase or tPA (10 mg / kg). IO@PDA + tPA + DNase appears to improve the recanalization at 24 hours but not as much as the IO @ PDA @tPA@ DNase-treated mice, for which a majority (75%) of complete recanalization was observed. D. Representative illustrations of the magnetic resonance angiography obtained for the mice depending on the treatment administered.

[0139] Figure 15. IO @ PDA @tPA@ DNase improves functional recovery. A. Schematic representation of the corridor test apparatus (left), and quantification of left-side object visits by mice treated with saline, IO@PDA, DNase, r-tPA (10 mg / kg), IO@PDA + r-tPA (2.5 mg / kg) + DNase, or IO @ PDA @tPA@ DNase. Assessments were performed before IS, and at 1 and 5 days post-IS (2-way ANOVA with multiple comparisons (n = 5-6 per group). B- D. Detailed analysis of the proportion of left-side object visits for each group at baseline (B), 1 day (C), and 5 days (D) after IS (n = 5-6 per group). E-G. Evaluation of motor and sensorimotor function using the corner test. Percentage of turns to the left or right before IS (E), at 1 day (F), and 5 days (G) post-IS (n = 5-6 per group).

[0140] EXAMPLES

[0141] Abbreviations

[0142] BSA: bovine serum albumin

[0143] CB V : cerebral blood volume

[0144] CLRmax: maximum clot lysis rate

[0145] CT: clotting time

[0146] DIV : days in vitro

[0147] DLS: dynamic light scattering

[0148] DMEM: Dulbecco's Modified Eagle medium

[0149] DNase: deoxyribonuclease

[0150] DWI: Diffusion- weighted images

[0151] ELS: electrophoretic light scattering fUS: functional ultrasound

[0152] HEPES: 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid

[0153] IO: ultrasmall particles of iron oxide

[0154] LT: lysis time

[0155] MCA: middle cerebral artery

[0156] MRI: magnetic resonance imaging MW: molecular weight

[0157] PBS: phosphate buffered saline

[0158] PDA: polydopamine

[0159] PEP: poly epinephrine

[0160] PNE: polynorepinephrine

[0161] PST: polyserotonine

[0162] RARE: rapid acquisition with relaxarion enhancement rtPA: recombinant tissue plasminogen activator

[0163] STZ: streptozotocin

[0164] TE: time of echo

[0165] TOF: time of flight tPA: tissue plasminogen activator

[0166] TR: repetition time

[0167] Materials and methods

[0168] Reagents

[0169] The following reagents were purchased from Sigma-Aldrich: ferric chloride hexahydrate, ferrous chloride tetrahydrate, ammonia solution, dopamine hydrochloride, sodium phosphate monobasic, sodium phosphate dibasic, mannitol. The tPA is obtained from dialyzed commercial recombinant tPA (Actilyse®, Boehringer Ingelheim). 1. Particles comprising ultrasmall particles of iron oxide embedded within poly dopamine (IO @ PDA)

[0170] 1.1. Synthesis of particles comprising ultrasmall particles of iron oxide embedded within polydopamine (IO @ PDA)

[0171] IO @ PDA were produced by a co-precipitation method in an alkaline buffer. In a typical synthesis, 540 mg of FeCh.bthO and 198.8 mg of FeChAHiO were dissolved in 5.7 mL of distilled water by vortexing, yielding a homogenous yellow solution. Under continuous agitation at room temperature, 6.3 mL of a 13% ammonia solution was progressively added at a rate of 0.2 mL / min. The solution turned from yellow to brown and, ultimately, to a deep black color, corresponding to the formation of magnetite. The precipitate was washed five times with distilled water by magnetic separation and resuspended in 10 mL of distilled water. Eight milliliters of the obtain solution was resuspended in 40 mL of a solution containing dopamine hydrochloride (2.5 mg / mL). The resulting solution was sonicated for 15 min at 70% amplitude and 26 kHz using a UP200ST sonicator (Hielscher). The color of the solution slightly changed from black to dark brown. Then, the solution was centrifuged at 3000g for 5 min to remove large remaining nanocrystals aggregates. Thirty milliliters of the supernatant containing IO @ Dopamine and free dopamine hydrochloride were transferred to a new vial. This vial was placed under vigorous steering using an Ultra-Turrax T-25 disperser at 20,500 rpm. To produce large IO@PDA, 67 pL of a 13% ammonia solution was first added to the solution, which was left to react for 60 min. Then, 203 pL of a 13% ammonia solution was added, and the incubation was continued for 30 min to allow further PDA. Then, the solution was centrifuged at 1000g for 3 min to remove the largest aggregates, the pellet was discarded, and 24 mL of the supernatant was transferred to a new vial. The IO @ PDA were then washed five times with distilled water and lastly resuspended in 8 mL of distilled water and stored at 4°C until further use.

[0172] 1.2. Determination of iron concentration

[0173] Iron content of IO @ PDA suspension was measured with the FerroZine method. Particles were degraded overnight at room temperature in 1 M HC1, releasing ferric (Fe3+) and ferrous (Fe2+) ions in solution. Samples were incubated 30 min with 0.65% (w / v) ascorbic acid to reduce ferric ions in ferrous ions. Sample pH was adjusted with 12% (w / v) ammonium acetate. 3-(2-Pyridyl)-5,6-diphenyl-l,2,4-triazine-p,p'-disulfonic acid monosodium salt hydrate was added (1 mM; FerroZine Iron Reagent, Sigma- Aldrich), and absorbance was measured at 562 nm with a spectrophotometer (ELx8O8 Absorbance reader, BioTek) indicating the amount of complexes formed with ferrous ions. Iron content was determined against standard curves obtained from iron chloride dilutions.

[0174] 1.3. Determination of the hydrodynamic diameter of the particles

[0175] Dynamic light scattering was used to determine the average hydrodynamic diameter, the poly dispersity index and the diameter distribution by volume of the IO @ PDA 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. All IO @ PDA bashed measure around 600 nm.

[0176] 2. Conjugate comprising the particle IO @ PDA and tPA (IO@PDA@tPA)

[0177] 2.1. Dialyzed tPA

[0178] Commercial recombinant tPA (Actilyse®, Boehringer Ingelheim) is reconstituted in ultra- pure water and transferred in a dialysis membrane (Spectra / Por® Dialyis Membrane, MWCO 12-14.000, TRIAL KITS). The dialysis membrane filed with tPA is placed under continuous agitation in HEPES buffer, pH 7.4 for 48 hours at 4°C. At the end, tPA concentration is determined with a Nanodrop (MW = 63 000 g.mol; a = 107620 l / g*cm)

[0179] 2.2. Conjugating tPA on IO @ PDA

[0180] 500 pL dialyzed tPA at 1.67 mg / mL in HEPES buffer, pH 7.4 are mixed with 500 pL of IO @ PDA in distilled water at 0.835 mg / mL iron concentration. This leads to a final concentration of 0.835 mg / mL tPA for 0.4175 mg / mL of IO.

[0181] The mixed solution is incubated at 4°C for 1 hour under constant rotation. After 1 h, a magnet is used to collect only the IO@PDA conjugated with tPA that will be resuspended in a 0.3 M mannitol solution. Before discarding the supernatant, a nanodrop is used to measure tPA concentration that does not fix on the particles (MW = 63 000 g.mol; a = 107620 l / g*cm).

[0182] 3 measures of the supernatant were performed. An average of these data gives the concentration of free tPA. Finally, by subtracting the initial concentration of added tPA, tPA concentration conjugated to IO @ PDA can be deduced. The actual final concentration of tPA attached to the IO@PDA varies between 0.3 mg / mL and 0.6 mg / mL. In this example, the deduced tPA concentration fixed on the particles is 0.4545 mg / mL. The concentration value obtained here was used to calculate the dose of IO@PDA@tPA to be injected in vivo.

[0183] 3. Verifying tPA activity and fibrinolytic action when linked to IO @ PDA

[0184] 3.1. Spectrofhior test

[0185] Several dilutions of tPA alone and IO@PDA@tPA in a Tris solution (50 mM Tris (pH 8.0) containing 150 mM NaCl) are made. Based on the datasheet, 500 ng, 250 ng and 100 ng are the most suitable. Then, the various samples are incubated (in triplicates) in the presence of a Anorogenic substrate (5 mM, Spcctrofluor FL444). The reaction will be carried out at 37°C in a total volume of 100 pL. The amidolytic activity will be measured as the change in Auorescence emission at 440 nm (excitation at 360 nm) over time with a multimode microplate reader (Spark®, TECAN). Activity of the tPA will be obtained from the initial slope regression (V0) and plotted over the initial protein mass added to the samples.

[0186] 3.2. Clot lysis test

[0187] The effect of IO@PDA@tPA during clot formation and lysis was studied by monitoring the change in turbidity in human plasma using a microplate reader (FLUOstar Optima, BMG Labtech). Calcium chloride (final concentration, 25 mM) was added to citrated plasma diluted 1:2 in HEPES buffer [10 mM HEPES, 150 mM NaCl, and 0.4% bovine serum albumin (BSA) (pH 7.4)] to promote coagulation. Samples were incubated at 37°C with IO @ PDA or tPA or IO@PDA@tPA or IO @ PDA + tPA at 1 and 5 nM tPA equivalence and absorbance (405 nm) was monitored for 12 hours every 30 s at 37 °C. Results are expressed as the time to achieve 75% maximal absorbance [clotting time (CT)], and the 50% lysis time was calculated as the time from initiation of clot formation to the time at which maximal absorbance falls to 50%. All experiments were performed in triplicate.

[0188] 3.3. Halo assay

[0189] Droplets of the clotting mixture (Innovin (Dade® Innovin®, Siemens, Munich, Germany), CaCl20.25 M, HEPES buffer (25 mM HEPES, 137 mM NaCl, pH 7.4)) are deposited on the bottom edge of the wells of a 96 well plate. The clotting mixture is spread around the edge of the wells with the tip of a P 100 micropipette containing 20 pL of blood. The blood is slowly released and mixed around the edge of the well thereby making use of the fluidic cohesion effect. After incubation at 37 °C for 30 minutes, the clots should have a homogenous halo shape at the bottom of the wells, leaving the centre area of the well clear and empty.

[0190] 2 conditions were tested: tPA and IO@PDA@tPA at 0.25; 0.1 and 0.075 mg / mL tPA equivalence). The fibrinolysis rate was assessed by one measurement at 510 nm every minute, with 5 seconds orbital shaking (200 rpm, 3 mm diameter) at each time point, over 2 hours at 37 °C with a multimode microplate reader (Spark®, TECAN). Negative controls for the assay were obtained from the addition of 75 pL of PBS to halo thrombi (no fibrinolytic drug), and positive controls consisted of a well containing 20 pL of blood and 80 pL of PBS (no clotting mixture). The positive control wells provided absorbance values corresponding to full degradation (Atotai), and the negative control wells provided reading for no degradation (Azero). At each time point, the percentage of degradation were obtained from this formula: Dx(t) = 100(Ax(t) - Azero(t)) / (Atotai(t) - Azero(t)). Finally, this experiment gave several data, the maximum degradation (Dmax), the activation time (At) and the maximum clot lysis rate (CLRmax).

[0191] 4. Conjugate comprising the particle IO @ PDA and urokinase

[0192] 4.1. Urokinase

[0193] Urokinase (THERASOLV 500 000 UI), a plasminogen activator, is used in case of thrombosis of central and peripheral venous catheters. 4.2. Conjugating urokinase on the IO @ PDA

[0194] 250 L urokinase (THERASOLV 500000 UI) at 1 mg / mL in NaCl buffer, pH 7.4 are mixed with 250 pL of IO @ PDA in distilled water at 0.5 mg / mL iron concentration. This leads to a final concentration of 0.5 mg / mL urokinase for 0.25 mg / mL of IO.

[0195] The mixed solution is incubated at 4°C for 1 hour under constant rotation. After 1 h, a magnet is used to collect only the IO @ PDA conjugated with urokinase that will be resuspended in a 0.3 M mannitol solution.

[0196] Before discarding the supernatant, a nanodrop is used to measure urokinase concentration that does not fix on the particles (MW = 46 383.7 g.mol; a = 72 320 l / g*cm).

[0197] 3 measures of the supernatant were performed. An average of these data gives the concentration of free urokinase. Linally, by subtracting the initial concentration of added urokinase, urokinase concentration conjugated to IO @ PDA can be deduced. The actual final concentration of urokinase attached to the IO@PDA was 0.2105 mg / mL.

[0198] 5. Conjugate comprising the particle IO @ PDA and tenecteplase (TNK)

[0199] 5.1. Tenecteplase

[0200] Tenecteplase (TNK), a recombinant form of tPA, is used as thrombolytic treatment indicated in adults in cases of suspected myocardial infarction. The tenecteplase was produced recombinantly from transfected HEK293 cells.

[0201] 5.2. Conjugating tenecteplase (TNK) on the IO @ PDA

[0202] 30 pL TNK at 1.3 mg / mL in HEPES buffer, pH 7.4 are mixed with 30 pL of IO @ PDA in distilled water at 0.5 mg / mL iron concentration. This leads to a final concentration of 0.65 mg / mL TNK for 0.25 mg / mL of IO.

[0203] The mixed solution is incubated at 4°C for 1 hour under constant rotation. After 1 h, a magnet is used to collect only the IO @ PDA conjugated with TNK that will be resuspended in a 0.3 M mannitol solution. Before discarding the supernatant, a nanodrop is used to measure TNK concentration that does not fix on the particles (MW = 59042.38 g.mol; a = 109 385 l / g*cm).

[0204] 3 measures of the supernatant were performed. An average of these data gives the concentration of free TNK. Finally, by subtracting the initial concentration of added TNK, TNK concentration conjugated to IO @ PDA can be deduced. The actual final concentration of TNK attached to the IO@PDA was 0.6142 mg / mL.

[0205] 5.3. Verifying urokinase and TNK activity when linked to IO @ PDA

[0206] A chromogenic assay was performed. In this assay, the chromogenic substrate S-2551 (Werfen, 00082033239) allows plasmin activity quantification. The reaction mixture contains Human glu-plasminogen (ERL, HPG 2001), either urokinase, TNK, tPA, IO @ PDA @ urokinase, IO @ PDA @ TNK or IO@PDA@tPA and S-2251. Plasmin generation at a given time concentration is linear, with a resulting acceleration of S-2251 hydrolysis. The amount of p-nitroaniline formed, and the absorbance of the solution at 405 nm, increases exponentially, and is proportional to the plasminogen activator concentration. The final concentrations of S-2251 is 0.6 mM, 0.25 pM for Human glu-plasminogen and different concentrations from 1; 2.5 and 5 nM for the plasminogen activators. All dilutions were made in a Tris 50 mM + NaCl 150 mM buffer at pH=8.0. After the preparation of the mix of plasminogen and plasminogen activators (Volume 50 pL), the reaction is initiated by the addition of the substrate solution at the end (volume 50 pL) to the wells. The final volume is 100 pL. The plate is read kinetically at 37 °C for up to 4 hours (taking readings every 30 seconds) at a wavelength of 405 nm.

[0207] 6. Conjugate comprising the particle IO@PDA@tPA and DNase 1 (Pulmozyne®)

[0208] The IO@PDA@tPA were synthesized as described before, except for the incubation time of the tPA wich was reduced to 30 min. After removing the supernatant and measure the tPA quantity fixed on the IO @ PDA, 500pl of DNAse 1 (1,17 mg / ml) was added to the particles and incubated 30 minutes at 4°C under constant agitation. After 30 minutes, a magnet is used to collect only the IO@PDA@tPA conjugated with DNase 1 that will be resuspended in a 0.3 M mannitol solution. Before discarding the supernatant, western blotting was used to dertemine the quantity of DNAse 1 fixed on the particles.

[0209] 6.1. Western Blotting

[0210] To determine the concentration of unbound DNase 1 in the sample, a Western blot was performed using a standard curve of increasing concentrations of free DNase I. Samples were prepared and loaded onto stain-free precast gels (10% Mini-PROTEAN® TGX™ Protein Gels, 4561031, Bio-Rad) along with the DNase 1 standards. Following electrophoresis, total protein content was visualized using the stain-free technology with the Bio-Rad ChemiDoc imaging system. Band intensity was quantified using Image Lab software (Bio-Rad), and the concentration of DNase 1 in the sample was interpolated from the standard curve based on densitometric analysis.

[0211] 6.2. Agarose gel electrophoresis for DNAse 1 activity assay

[0212] To assess the nuclease activity of free and particle-bound DNasel, DNA degradation assays were performed using salmon sperm DNA (Invitrogen, ThermoFisher Scientific, 15632- 011) as a substrate. DNA samples (0,1 pg / p L) were incubated at 37 °C for 30 minutes under the following conditions: (1) DNA alone (negative control), (2) DNA with free DNase I (1 pg or 2 pg), (3) DNA with IO@PDA particles (particle control), and (4) DNA with IO @ PDA @tPA@ DNase particles (1 pg or 2 pg). The final volume of each reaction was adjusted to 15 pL with nuclease-free water.

[0213] Following incubation, samples were mixed with loading buffer and loaded onto a 1% agarose gel containing a nucleic acid stain (GelRed Nucleic Acid Gel Stain, Fisher Scientific, NC9938951). Electrophoresis was carried out at 100 V for 30-40 minutes in lx TAE buffer. DNA bands were visualized using a UV transilluminator or gel imaging system (Bio-Rad). The degradation of salmon DNA was assessed qualitatively by comparing the intensity and pattern of DNA bands across conditions, confirming whether the enzyme retained its activity when conjugated to particles.

[0214] 6.3. Clot degradation assay Human thrombi were obtained post-thrombectomy from stroke patients and stored in the Franch National COMPOCLOT biobank. Each clot was bisected to allow comparison of two treatment conditions using matched clot material. Clot halves were transferred into individual wells of a 24- well plate and incubated in 150 pL of BAPA plasma (Hirudin-based anticoagulated plasma) for 1 hour at 37 °C with gentle shaking (400 RPM) to allow rehydration. After rehydration, clots were gently blotted to remove excess fluid and weighed individually using an analytical balance to determine baseline mass. Treatments were then applied directly onto the clots. Treatment solutions were prepared in HBSS (Hank's Balanced Salt Solution) containing Ca2+and Mg2+, supplemented with BAPA plasma. The total volume of treatment applied to each clot was adjusted based on clot weight, using a ratio of 40 pL per mg of clot. Final concentrations were 1 pg / mL for tissue plasminogen activator (tPA) and 0.6 pg / mL for DNase 1 when applicable. Plates were incubated at 37 °C with shaking (400 RPM) for 2 hours. Clots were removed and weighed every 20 minutes to monitor time-dependent degradation. Three pairwise comparisons were performed:

[0215] - tPA vs IO @ PDA @ tPA; tPA + DNase 1 vs IO @ PDA @tPA@ DNase;

[0216] - IO@PDA@tPA vs IO @ PDA @tPA@ DNase.

[0217] Each clot was used for a single treatment comparison, ensuring intra-clot consistency between conditions.

[0218] 7. In vivo experiments

[0219] 7.1. Animals

[0220] All experiments were conducted in compliance with French ethical law (Decrees 2013-118 and 2020-274) and the European Communities Council guidelines (2010 / 63 / EU). Experiments were approved by the local ethical committee of Normandy (CENOMEXA, APAFIS#36663 and #45085). Animals were provided and maintained under specific pathogen-free conditions at the Centre Universitaire de Ressources Biologiques (CURB, Basse-Normandie, France), and all had free access to food and tap water. Mice were housed in a temperature-controlled room on a 12-hour light / 12-hour dark cycle with food and water ad libitum. A catheter was inserted into the tail vein of the mice for intravenous administration of the conjugate or treatments before the thrombin model and MRI acquisition. After surgery, animals were allowed to recover in a clean heated cage before taking them back to the animal facility.

[0221] 7.2. Hyperglycemic mice model induced by streptozotocin (STZ)

[0222] STZ (S0130_500MG, Sigma- Aldrich) was injected intraperitoneally to the mice during five days at 40 mg / kg. Blood sugar was monitored at 1-day pre-treatment, 1 day, 2 days, 8 days, 14 days and 21 days post treatment after 4 hours of fasting. On day 21, mice were included in the study if the blood sugar was above 200 mg / dl.

[0223] 7.3. Thrombin model

[0224] Before the surgical procedure, a pipette was made with hematologic micropipettes (calibrated at 15 mm / L; Assistent ref. 555 / 5; Hoecht, Sondheim-Rhoen, Germany) by using an electrophysiology puller (PC- 10; Narishige). Thereafter, the micropipette was pneumatically filled with 1 p L of purified murine alpha-thrombin (approximately 1000 NIH units / mg; Sigma-Aldrich) by applying negative pressure. Mice were anesthetized with isoflurane (5%, 70 / 30 NO2 / O2). Mice were placed in a stereotaxic device and maintained under anaesthesia with isoflurane (1.5-2%, 70 / 30 NO2 / O2) at 37°C by the integrated heat animal holder. Before beginning the surgery, buprenorphine (BUPRECARE, H0270. 54000561, 0.3 mg / mL) was injected to the mice for analgesia. Then, the skin between the right eye and the right ear was incised, and the temporal muscle was retracted. A small craniotomy was performed, the dura was excised, and the middle cerebral artery (MCA) was exposed. The pipette was introduced into the lumen of the MCA and 1 p L of purified murine alpha-thrombin (0.75 UI) was pneumatically injected (by applying positive pressure with a syringe connected to the pipette through a catheter) to induce the formation of a clot in situ. The pipette was removed 5 minutes after the injection of alpha thrombin at which time the clot had stabilized.

[0225] 7.4. AlCh model

[0226] Mice were anesthetized with isoflurane (5%, 70 / 30 NO2 / O2). Mice were placed in a stereotaxic device and maintained under anaesthesia with isoflurane ( 1.5-2%, 70 / 30 NO2 / O2) at 37 °C by the integrated heat animal holder. Before beginning the surgery, buprenorphine (BUPRECARE, H0270. 54000561, 0.3mg / ml) was injected to the mice for analgesia. The MCA was exposed and a piece of AlCh (Sigma- Aldrich) -saturated filter paper was topically applied on the artery for 5 minutes.

[0227] 7.5. IO @ PDA @ tPA preparation and injection

[0228] As described in paragraph 2.2. above, tPA is conjugated to the particles 24 hours before inducing the thrombin stroke model on mice. Based on the deduced tPA concentration conjugated to IO@PDA (which varies between 0.3 and 0.6 mg / mL, in this example it was 0.4545 mg / mL) and the animal weight, the intravenous injection volume of IO@PDA@tPA in mice at the desired doses of tPA is determined. For example, to inject 2.5 mg / kg tPA equivalence of IO@PDA@tPA to a 40 g mouse: 2.5 (concentration desired / (0.4545 (tPA concentration fixed on the particles) x 40 (mouse weight)) = 137.5 pL.

[0229] 7.6. MRI acquisition

[0230] Experiments were performed using a BioSpec 7-T TEP-MRI system with a volume coil resonator (Bruker, 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.

[0231] Brain scans including an altemance of T2*-weighted sequence for iron- sensitive imaging with TR / TE 50 ms / 8.6 ms and TOF sequence to visualize vascular structure, with TR / TE = 12 ms / 4.2 ms were made 20 min after treatments injection: IO @ PDA (2 mg / kg); IO@PDA@tPA at 2.5 mg / kg tPA and 2 mg / kg IO @ PDA equivalence; IO@PDA@tPA at 5 mg / kg tPA and 4 mg / kg IO @ PDA equivalence; IO @ PDA (2 mg / kg) + tPA (2.5 mg / kg) for 45 minutes.

[0232] Diffusion-weighted images (DWI) were acquired using a standard spin echo imaging modified with a Stejskal-Tanner gradient scheme (TE / TR 38 ms / 2000 ms, with 75 x 75 x 500 pm3spatial resolution, giving an in-plane resolution of 100 x 78 mm, slice thickness of 0.75 mm, one direction diffusion gradient, in the frequency encoding direction) with a b factor of 1000 s / mm2and one averaging. Then brain scans including of T2-weighted (RARE sequence, with TR / TE = 3500 ms / 40 ms), T2*-weighted sequences (fast-low angle shot (FLASH) sequence, with TR / TE = 50 ms / 3.5 ms) and TOF sequences (TR / TE = 12 ms / 4.2 ms) were made 24 hours after treatments injection to visualize the lesion size, the recanalization and haemorrhagic transformation.

[0233] Signal void quantification on 3D T2* -weighted images, and 3D representation of IO @ PDA or IO@PDA@tPA induced hyposignal were realised using the segmentation and thresholding module on Slicer software (v4.11). Results are presented as volume of particles- induced signal void, or area of signal void (in mm3).

[0234] 7.7. Ultrasound (fUS) imaging

[0235] 7.8. All ultrasound imaging acquisitions were performed from bregma -3 mm to bregma +1 mm, both before and 15 minutes after stroke onset, using an ultrafast scanner (Iconeus One, Iconeus, France), the IcoScan acquisition software, and a dedicated ultrasound probe (Iconeus IcoPrime-4D MultiArray, 15 MHz, 256 elements, 100 pm pitch). Mice were placed in a stereotaxic frame. After incision of the scalp over the skull, both temporal crests were thinned to facilitate ultrasound wave penetration into the cortex and to prevent resolution loss. A cranial window was performed only on the left side to access the MCA (to avoid signal variations due to drilling). The skull was then cleaned with saline. Centrifuged ultrasound gel (2,500 x g, 5 min) was applied to the skull before placing the probe. CBV monitoring: A 5-minute acquisition was performed before stroke induction to obtain baseline CBV (TO). A 45-minute acquisition was then conducted 15 minutes after stroke onset to monitor CBV variations. Five minutes after the start of the 45-minute acquisition (i.e., 20 minutes after stroke onset), mice received one of the following treatments: IO@PDA (2 mg.kg); IO @ PDA @tPA@ DNase (2.5 mg / kg tPA equivalency and 1.32 mg / kg DNase equivalency); IO@PDA (2 mg / kg) + tPA (2.5 mg / kg) + DNAse (1.32 mg / kg); DNase (1.32 mg / kg) or tPA (10 mg / kg)). CBV variations: Two brain regions were selected for analysis: the right isocortex corresponding to the contralateral hemisphere and the ipsilateral hemisphere corresponding to the lesion site. Average CBV in each area was calculated over the 5-minute baseline acquisition and at each 5-minute interval during the post-ischemic acquisition. To evaluate reperfusion in the ipsilateral cortex following treatment, CBV was further analysed every 10 minutes. CBV variations were expressed as the percentage change relative to the baseline CBV. Grip test

[0236] Functional recovery was assesed by measuring the animal’s forepaws strength with a BIO- GS3 Grip Strength Test (Bioseb). Comparison between the baseline (day before stroke) with 1 day, and 5 days after stroke for both forepaws and a ratio between the strength of left forepaw and the right forepaw was made.

[0237] 7.9. Corridor test

[0238] To assess sensorimotor function, we designed a custom-made corridor task, based on the ability of animals to explore objects. The apparatus consisted of a black PVC squared corridor (120 cm long, 6 cm wide, 16 cm wall’s height). On each wall (left and right) four objects (custom-made, 3D-printed, 2 cm height, 2 width, 1 cm depth), spaced 16.5 cm apart, were fixed at 1 cm from the ground. Shape and colour of the objects were changed on each test day. Mice were habituated for 1 hour in the experiment room. Then each mouse was placed at the beginning of the corridor, the head facing the end, and allowed to cross freely through the corridor during 1 min period. Exploration of the objects was quantified on each side of the corridor.

[0239] 7.10. Corner test

[0240] The corner test was used to assess sensorimotor and post-stroke asymmetry. Mice were placed between two angled vertical boards forming a 30° comer. When entering the comer, mice typically rear and turn either to the left or the right to exit. Each mouse underwent 10 consecutive trials per session. The direction of turning (left or right) was recorded for each trial. A preference for turning contralateral to the lesion side was interpreted as sensorimotor impairment. The test was conducted before, 24h and 5 days after stroke onset. Results are presented as the percentage of turns toward each side, indicating turning side preference.

[0241] 8. Statistical analysis All results are presented as mean ± Standard Deviation (SD). Statistical analyses were performed blinded to the experimental groups, using Graph Pad Prism software (v8.0). We assumed normality of the data distribution with Shapiro-Wilk test. 2- way ANOVA, Kruskal- Wallis with Tukey’s multiple comparisons test was used for comparing more than two groups. Differences were considered statistically significant if probability value p < 0.05.

[0242] 9. Acceptance Criteria

[0243] Mice were excluded in case of death during the experimental procedure, technical problems, or haemorrhage, or if mice presented a lesion size superior to 35 mm2or inferior to 10 mm2(without injection of a thrombolytic agent).

[0244] Results

[0245] Iron oxide nanoparticles (IO) were synthetized via a simple co-precipitation of Fe2+ / Fe3+ions, leading to nanocrystal formation. These nanocrystals were first coated with dopamine. Then, the polymerization of dopamine was induced in an alkaline buffer triggering the formation of a polydopamine matrix with trapped nanocrystals (IO@PDA; Figure 1A & Figure 6A). The resulting IO@PDA particles had a hydrodynamic diameter of 614.6 nm ± 74.5 nm and a zeta potential of -23.79 mV ± 3.6 mV (Figure IB & Figure 6B). Subsequently, a defined concentration of dialyzed recombinant tissue-type plasminogen activator (tPA) was added to the IO @ PDA batch and incubated for 1 hour at 4°C under constant rotation, allowing the tPA to graft onto the particle surfaces (Figure 1A & Figure 6A). The average diameter of the IO@PDA@tPA is slightly modified, a slight increase in particle size dispersion is observed, with no visible aggregation. Moreover, they retained the same zeta potential as the unmodified IO@PDA (Figure 6B,D). Transmission electron microscopy (TEM) of the IO@PDA@tPA revealed a surrounding veil structure (Figure 6C). Additionally, confocal microscopy using tPA conjugated with Alexa Fluor 488 confirmed successful tPA functionalization on the IO@PDA surface (Figure 6D).

[0246] Spectrofluor test demonstrates that tPA activity remains intact after binding on IO @ PDA (Figure ID). The plasma clot lysis assay (platelets poor clots) shows that the IO@PDA@tPA does not interfer with the clot formation (Figure 2B) and that the thrombolytics capacities of the IO@PDA@tPA are equivalent to tPA alone (Figure 2C), which is confirmed with the whole blood halo assay (platelets rich clot; figure 2D,E).

[0247] Early experiments revealed that activity levels in the spectrofluorimetric assay correlates to the respective tPA concentration (Figure 7 A). The use of non-dialyzed tPA containing large amounts of arginine as an excipient adversely affected its amidolytic activity when grafted onto IO@PDA (Figure 7C,D), probably by interfering with tPA binding. Of note, buffer composition of the suspension of IO@PDA@tPA also influenced tPA activity, with HEPES buffer negatively affecting its amidolytic activity (Figure 7E,F). This led to the use of 0.3 M mannitol in the IO@PDA@tPA suspension. Furthermore, the tPA concentration must be at least twice that of the IO @ PDA concentration (in mass) to reach a detectable amidolytic activity of the final particles (Figure 7G,H).

[0248] In vivo experiments reveal that the hyposignal for microthrombi quantification is decreased after injection of IO@PDA@tPA compared to IO @ PDA during the 1 h MRI acquisition after inducing the thrombin stroke model (Figure 3B). Re-injection of IO @ PDA alone after IO@PDA@tPA treatment in MRI confirms that the loss of the hypotense signal corresponding to microthrombi is due to their lysis by the IO@PDA@tPA and not because of a non-targeting of the microthrombi by the particles (Figure 3C).

[0249] More, the lesion size at 24 h is significantly decreased in the IO@PDA@tPA 2.5 mg / kg condition compared to the other groups (Figure 4B). Angiographies reveal a majority of no recanalization in the IO@PDA and IO@PDA + tPA 2.5 mg / kg condition but 75% and 68% of complete recanalization at 24 h after IO@PDA@tPA 2.5 mg / kg and 5 mg / kg administration respectively (Figure 4C). Interestingly, the IO@PDA@tPA 5 mg / kg condition has no impact on the lesion size (no differences with the IO @ PDA and IO@PDA + tPA 2.5 mg / kg; Figure 4B) whereas it induces a decrease of the microthrombi hyposignal on MRI (Figure 3B) and a majority of complete recanalization (Figure 4C). Finally, mice treated with IO@PDA@tPA present a full functional recovery at 5 days after stroke whereas the mice treated with IO @ PDA or IO@PDA + tPA 2.5 mg / kg still have functional impairments (Figure 4D). Two other conjugates comprising tenecteplase (TNK) or urokinase as plasminogen activators were also prepared (Figure 5A) and tested. The chromogenic assay demonstrates that the IO@PDA linked with tPA (figure 5B), Urokinase (figure 5C), and TNK (figure 5D) possess the same activity properties as if they were alone in the solution.

[0250] Another sert of in vivo experiments were performed on hyperglycemic mice: induced by repetead injection of STZ (40mg / KG) for five days. After inducing ischemic stroke, lesion size, recanalization, hemorrhagic transformation, and functional recovery were assessed, depending on the treatment received (IO@PDA, IO@PDA@tPA 2.5 mg / kg, IO@PDA + tPA 2.5 mg / kg, tPA 10 mg / kg, or IO@PDA + tPA 10 mg / kg; Figure 8A). The experiments revealed that the mean lesion size at 24 h is significantly decreased in the IO@PDA@tPA 2.5 mg / kg group compared to the IO@PDA-treated mice. Other groups showed reduced but not significant lesion size (Figure 8B). Angiographies revealed that most mice in the IO@PDA group showed no recanalization, while 18%, 22%, and 20% of no recanalization events were observed for the IO@PDA + tPA 2.5 mg / kg; IO@PDA + tPA 10 mg / kg and tPA 10 mg / kg groups, respectively, at 24h after stroke (Figure 8C). In contrast, IO@PDA@tPA 2.5 mg / kg treated mice showed 36% of complete recanalization at 24h after stroke onset (Figure 8C). At 5 days after stroke, 63% of the IO@PDA@tPA 2.5 mg / kg treated mice had a complete recanalization and 37% showed partial recanalization (Figure 8D). Mice treated with tPA 10 mg / kg showed only 27% of complete recanalization and 73% of partial recanalization. For the other groups we observed the same percentage of partial recanalization as in the mice treated with tPA 10 mg / kg but 9 to 20% of them didn’t show any recanalization (Figure 8D). T2s-weighted MRI acquisition revealed no hemorrhagic transformation 24 hours after stroke (Figure 9). However, hypointense signals corresponding to micro thrombi were observed in the IO @ PDA and the IO @ PDA + tPA 2.5 mg / kg treated mice confirming that without tPA or if tPA is not linked to IO @ PDA at a lower dose, microthrombi are not completely lysed (Figure 9). At 5 days after stroke, IO@PDA@tPA 2.5 mg / kg treated mice showed no hemorrhagic transformation (67%) and 33% of small petechiae whereas, 9 to 22% of the mice treated with the other groups exhibited hemorrhagic transformation (Figure 8F). Functional recovery was investigated by evaluating the global strength of the forepaw and the specific strength of the left paw with a grip test as well as sensorimotor function by a custom-made corridor task, based on the ability of animals to explore objects (Figure 8G,H and Figure 10). The grip test results revealed that the IO @ PDA and tPA 10 mg / kg treated mice have a global strength deficit at 1 day and 5 days after stroke (Figure 8G). The IO @ PDA + tPA 10 mg / kg treated mice had a global strength deficit at 1 day after stroke (Figure 8G); and the IO@PDA@tPA 2.5 mg / kg and IO@PDA + tPA 2.5 mg / kg treated mice had no global strength deficit at 1 and 5 days after stroke (Figure 8G). Regarding the left paw strength, IO @ PDA treated mice presented deficit at 1 day and 5 days after stroke which was, however, not significant (Figure 8H); the IO@PDA + tPA 10 mg / kg treated mice had strength deficit at 1 day and 5 days after stroke but started recovering after 5 days (Figure 8H); the tPA lOmg / kg treated mice had strength deficit at 1 day after stroke but no deficit at 5 days after stroke (Figure 8H); the IO@PDA@tPA 2.5 mg / kg and the IO@PDA + tPA 2.5 mg / kg treated mice had strength deficit at 1 day after stroke but no deficit at 5 days after stroke (Figure 8H). For the sensorimotor function with the corridor test, all groups displayed a deficit on the number of visited objects at 1 day after stroke (Figure 10). The IO@PDA; IO @ PDA + tPA 10 mg / kg; tPA 10 mg / kg treated mice still had a deficit on the number of visited objects at 5 days after stroke (Figure 10). The IO@PDA + tPA 2.5 mg / kg and the IO@PDA@tPA 2.5mg / kg treated mice no longer had a deficit at 5 days after stroke (Figure 10).

[0251] 20% to 30% of ischemic clots are tPA-resistant. Thus, a new conjugate was designed, the IO @ PDA @tPA@ DNase. The synthesis of the new conjugate is the same as described above for the IO @ PDA formation, and the conjugation of tPA and then DNase is essentially the same (Figure 11 A). The amount of tPA grafted on the surface of the IO @ PDA remains the same as previously described (Figure 1 IB), and the quantity of DNase conjugated to the particles is around 0.3 mg / ml (Figure 1 IB). DNA electrophoresis for DNase 1 activity verification revealed that the DNase enzymatic properties remain intact even when grafted to the IO@PDA@tPA (Figure 11C). Finally, the Spectrofluor test, as described above (Figure 1C,D & Figure 7A), demonstrated that the amydolytic capacities of tPA are not influenced by the addition of DNase to the conjugate (Figure 1 ID, E, F).The new conjugate, IO @ PDA @tPA@ DNAse, was tested in vitro using human thrombi obtained post- thrombectomy from IS stroke patients (Figure 12A). Each clot was bisected to allow comparison between two treatment conditions using matched clot material (Figure 12A). Over two hours, clots were incubated with tPA vs IO@PDA@tPA (Figure 12B); tPA & DNAse vs IO @ PDA @tPA@ DNAse (Figure 12C); or IO@PDA@tPA vs IO @ PDA @tPA@ DNase, allowing pairwise comparison (Figure 12D). Quantification revealed no significant differences between the percentage of lysis for clots treated with tPA and IO@PDA@tPA (Figure 12E). The same was observed for clots treated with tPA & DNase vs IO @ PDA @tPA@ DNase (Figure 12F). However, the mean lysis percentage was higher, around 20% (Figure 12F), compared to the previous conditions (around 9%, Figure HE), already highlighting the beneficial role of DNase on tPA-resistant thrombi. Finally, clots treated with IO @ PDA @tPA@ DNase were significantly more lysed, around 35%, compared to clots treated with IO@PDA@tPA (around 15%, Figure 12G), demonstrating the beneficial added effect of DNase to the conjugate.

[0252] The therapeutic potential of the new conjugate was next assessed in vivo. The AlCh-induced IS model was selected due to its ability to generate r-tPA-resistant clots. Notably, this model does not induce microthrombi formation in the microvasculature, precluding the use of molecular MRI for early-phase monitoring. To overcome this limitation, functional ultrasound (fUS) imaging was employed to assess cerebral blood volume (CBV) before and during the acute phase of IS (Figure 13A). Five treatments groups were evaluated (Figure 13B-G): IO@PDA (2mg / kg); DNase (1.37 mg / kg); tPA (10 mg / kg); a combination of IO@PDA (2 mg / kg) + tPA (2.5 mg / kg) + DNase (1.37 mg / kg) and the IO @ PDA @tPA@ DNase conjugate (corresponding to 2.5 mg / kg tPA and 1.37 mg / kg DNase equivalency). In mice treated with IO@PDA (Figure 13B), DNase (Figure 13C), tPA (Figure 13D) and IO@PDA + tPA + DNase (Figure 13E), the CBV dropped significantly — by approximately 60 to 80 % and remain decreased (2-way ANOVA, Multiple comparisons, *** p < 0.001 and **** p < 0.0001). For mice treated with IO @ PDA @tPA@ DNase, mice have indeed a significant drop of CBV compared to baseline after IS as expected, however a rapid and progressive reperfusion was observed starting 10 minutes after treatment injection (Figure 13F, G) indicating effective revascularization. At 24 hours post-ischemic stroke, mice treated with IO @ PDA @tPA@ DNase exhibited a homogeneous reduction in lesion size compared to the other treatment groups (Figure 14A, B). Interestingly, tPA at 10 mg / kg appeared to reduce the ischemic area to a similar extent as IO @ PDA @tPA@ DNase (Figure 14A, B), despite a continuous drop in CBV during the acute phase of IS, which typically predicts a larger infarct volume (Figure 14D). Angiographic analysis performed 24 hours after IS revealed persistent occlusion in 75%, 50%, 71%, and 100% of mice treated with Saline, IO@PDA, DNase, and tPA, respectively. In contrast, 75% of mice treated with IO @ PDA @tPA@ DNase showed complete recanalization, further supporting the therapeutic efficacy of the conjugate (Figure 14C, D). Notably, the combined administration of IO @ PDA + tPA + DNase also improved recanalization rates compared to single-agent treatments (Figure 14C, D).

[0253] Finally, to assess functional recovery following ischemic stroke, mice were subjected to the corridor test prior to IS induction, and at 1- and 5-days post-IS. Mice treated with Saline, IO @ PDA, DNase, tPA, or the combination of IO @ PDA + tPA + DNase showed impaired exploratory behavior at both day 1 and day 5 (Figure 15A-D). In parallel, sensorimotor performance was assessed using the Comer test at baseline, and at 1 and 5 days following ischemic stroke (Figure 15E-G). At baseline, several groups (including Saline, IO @ PDA, and tPA) already exhibited a moderate right-turning bias. Conversely, DNase, IO@PDA + tPA + DNase, and IO @ PDA @tPA@ DNase groups showed more balanced behavior (Figure 15E). At 24 hours post-IS, all groups exhibited a marked rightward turning bias, reflecting acute sensorimotor deficits (Figure 15F). Interestingly, only IO @ PDA @tPA@ DNase- treated mice maintained a symmetrical turning behavior, suggesting early functional recovery. By 5 days post-IS, most groups (Saline, IO@PDA, and IO @ PDA @tPA@ DNase) displayed near- symmetric turning patterns, indicating spontaneous or treatment-facilitated recovery (Figure 15G). In contrast, mice treated with DNase alone continued to exhibit a strong right-turning bias, and those treated with tPA or the IO @ PDA + tPA + DNase combination showed only partial improvement. These results further highlight the superior and sustained efficacy of the IO @ PDA @tPA@ DNase conjugate in promoting functional recovery

Claims

CLAIMS1. A conjugate comprising a suspension of particles and an activator of fibrinolysis or a fragment thereof, wherein the suspension of particles comprises particles having a hydrodynamic diameter comprised between 200 and 2000 nm, said particles comprising ultrasmall particles of iron oxide having a diameter between 1 and 50 nm embedded within a polymer matrix selected from polycathecolamines or polyserotonine.

2. The conjugate according to claim 1, wherein the iron oxide is selected from FciOa, FeaCU, or a mixture of FC2O3 and FcaCC3. The conjugate according to claim 1 or 2, wherein the polymer matrix is selected from polydopamine (PDA), polynorepinephrine (PNE), polyepinephrine (PEP) and polyserotonine (PST).

4. The conjugate according to any one of claims 1 to 3, wherein the iron concentration in the particles of the suspension of particles is comprised between 50% and 95% in weight with respect to the total weight of the particles.

5. The conjugate according to any one of claims 1 to 4, wherein the suspension of particles comprises a solvent selected from an aqueous solution, a saline solution, a glycerol solution and a mannitol solution.

6. The conjugate according to any one of claims 1 to 5, wherein the activator of fibrinolysis is a tissue plasminogen activator (tPA), preferably the activator of fibrinolysis is a is a recombinant tissue plasminogen activator (rtPA).

7. The conjugate according to claim 6, wherein the recombinant tissue plasminogen activator is alteplase, reteplase, urokinase, tenecteplase, desmatoplase, streptokinase or optimized tPA (OptPA).

8. The conjugate according to any one of claims 1 to 7, wherein the amount of particles is comprised between 10% and 35% in weight with respect to the total weight of the conjugate.

9. The conjugate according to any one of claims 1 to 8, further comprising a deoxyribonuclease.

10. The conjugate according to claim 9, wherein the amount of deoxyribonuclease is comprised between 15% and 40% in weight with respect to the total weight of the conjugate.

11. A suspension comprising the conjugate according to any one of claims 1 to 10 and a physiological medium.

12. The suspension according to claim 11, wherein the physiological medium is mannitol or glycerol.

13. The suspension according to claim 11 or 12, wherein the concentration of tissue plasminogen activator is comprised between 0.3 mg / mL and 0.6 mg / mL.

14. The conjugate according to any one of claims 1 to 10 or the suspension according to any one of claims 11 to 13, for use in an in vivo method of detection of microthrombi in a condition requiring thrombolysis, comprising the steps of: a) administering to a patient the conjugate according to any one of claims 1 to 10 or the suspension according to any one of claims 11 to 13; b) detecting the microthrombi by magnetic resonance imaging (MRI).

15. The conjugate for use according to claim 14 or the suspension for use according to claim 14, wherein the conjugate is administered to the patient in an amount comprised between 0.1 mg / kg and 1.0 mg / kg, with respect to to the weight of the patient.

16. The conjugate according to any one of claims 1 to 10 or the suspension according to any one of claims 11 to 13, for use in the treatment of a condition requiring thrombolysis, preferably the condition requiring thrombolysis is selected from acute ischemic stroke, myocardial infarction, pulmonary embolism, deep vein thrombosis,renal vein thrombosis, carotid thrombosis, portal vein thrombosis, mesenteric vein thrombosis, and local fibrinolytic therapy in intracranial hemorrhages.

17. The conjugate for use according to claim 16 or the suspension for use according to claim 16, wherein the treatment of a condition requiring thrombolysis is monitored by magnetic resonance imaging (MRI).

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