Hybrid particles comprising a cross-linked polysaccharide matrix and metal oxide nanoparticles

Abstract

Hybrid particles comprising a cross-linked polysaccharide matrix and metal oxide nanoparticles The invention relates to a method of manufacturing hybrid particles comprising a matrix of cross-linked polysaccharide and metal oxide nanoparticles embedded therein. It has been found that hybrid particles with low polydispersity and a high load of metal oxide nanoparticles could be obtained when non-coated metal oxide nanoparticles are used. Aggregation of the non-coated metal oxide nanoparticles could be controlled to achieve said polydispersity and load. The hybrid particles of the invention, when the metal oxide is a magnetic and photothermal metal oxide, could act on the thrombosis via a combination of thermal and mechanical action, through light and magnetic stimulation, and would be targeted to the occluded vessel by an external permanent magnet.

Description

[0001] HYBRID PARTICLES COMPRISING A CROSS-LINKED POLYSACCHARIDE MATRIX AND METAL OXIDE

[0002] NANOPARTICLES

[0003] Technical domain

[0004] The present disclosure relates to cross-linked polysaccharide particles comprising metal oxide (hybrid particles) for applications in the medical field. The present disclosure also relates to the preparation of such hybrid particles.

[0005] Background

[0006] Thrombosis is responsible for most strokes and heart attacks, which are the two leading causes of death worldwide. Reperfusion is commonly performed with thrombolytics such as recombinant tissue plasminogen activator (rt-PA) but these present numerous limitations (severe adverse effects, few eligible patients, low recanalization rate).

[0007] With the objective of designing a safer and efficient treatment, non-pharmaceutical treatments of thrombosis (i.e. targeted physical action on the thrombus) have been proposed.

[0008] In particular, Liu et al. (Liu C.-H., Liu M.-C., Jheng P.-R., Yu J., Fan Y.-J., Liang J.-W., Hsiao Y.-C., Chiang C.- W., Bolouki N., Lee J.-W., Hsieh J.-H., Mansel B. W., Chen Y.-T., Nguyen H. T., Chuang E.- Y. Plasma-Derived Nanoclusters for Site-Specific Multimodality Photo / Magnetic Thrombus Theranostics. Adv Healthcare Mater, 2301504 (2023)) have disclosed magnetic iron oxide nanoclusters that would act on the thrombosis via a combination of thermal and mechanical action, through light and magnetic stimulation, and would be targeted to the occluded vessel by an external permanent magnet.

[0009] However, the particles of Liu et al. remain very small (about 200 nm in diameter) and therefore are expected to have only a limited efficacy when exerting a mechanical action. Larger particles with a larger load of the magnetic oxide are expected to be more efficient for mechanical action and to keep their photothermal properties.

[0010] Previous research performed by the inventors, published in Bonnard et al. (Bonnard T., Serfaty J.M., Journe C., Ho Tin Noe B., Arnaud D., Louedec L., Derkaoui S.M., Letourneur D., Chauvierre C., Le Visage C. Leukocyte mimetic polysaccharide microparticles tracked in vivo on activated endothelium and in abdominal aortic aneurysm. Acta Biomater 10, 8, 3535-3545 (2014)) showed that it was possible to load particles comprising a cross-linked polymeric matrix with ultra-small iron oxide particles (USPIOs). However, the particles obtained had a high polydispersity and a limited load in USPIOs, which was sufficient since the aim was to image the thrombus and not to treat it. In Bonnard et al., dextran coated USPIOs were used. The choice of dextran coated USPIOs was motivated by two ideas:

[0011] It would help obtain an efficient loading of the USPIOs in the polysaccharide matrix because the dextran coating the USPIOs would cross-link with the dextran in the polysaccharide matrix, It would prevent uncontrolled aggregation of the USPIOs in the alkaline conditions required for the cross-linking of the polysaccharide.

[0012] Surprisingly, by going against this prejudice and by using non-coated USPIOs, the inventors have observed that it was possible to obtain particles comprising a cross-linked polymeric matrix and USPIOs. Moreover, the obtained particles had higher loading in metal oxide and a lower polydispersity than the particles of Bonnard et al. This is the object of the present disclosure.

[0013] Summary

[0014] The present invention relates to the following items:

[0015] Item 1 : A method for the preparation of particles A, said particles A comprising a cross-linked polysaccharide matrix and metal oxide nanoparticles B, said method comprising the following steps: a) providing an aqueous solution comprising the polysaccharide; b) providing an aqueous suspension of the nanoparticles B; c) providing an oil phase; d) mixing said aqueous solution and said aqueous suspension to obtain an aqueous mix; e) adding a base to the aqueous mix; f) adding a cross-linking agent to the aqueous mix; g) dispersing the aqueous mix in the oil phase in presence of a surfactant to obtain a water / oil (w / o) emulsion; h) crosslinking the polysaccharide to obtain the particles A, wherein the crosslinking comprises the reaction between a crosslinking agent and the polysaccharide to obtain the cross-linked polysaccharide matrix comprised in the particles A; i) recovering the particles A; wherein steps d) to i) are performed successively in this order and wherein the nanoparticles B introduced at step d) are non-coated metal oxide nanoparticles.

[0016] Item 2: the method of item 1 , wherein the nanoparticles B have a size of between 1 .5 nm and 50 nm, for example of 5 nm to 25 nm, as measured by transmission electron microscopy (TEM).

[0017] Item 3: the method of item 1 or 2, wherein the nanoparticles B are non-core-shell nanoparticles.

[0018] Item 4: the method of any one of items 1 to 3, wherein the nanoparticles B consist of the metal oxide. Item 5: the method of any one of items 1 to 4, wherein the metal oxide is a magnetic oxide.

[0019] Item 6: the method of item 5, wherein the magnetic oxide is ferrimagnetic or ferromagnetic.

[0020] Item 7: the method of item 5 or 6, wherein the nanoparticles B have a magnetization per unit of mass of over 20 x 10-3A.m2 / gram.

[0021] Item 8: the method of any one of items 1 to 7, wherein the metal oxide is an oxide of a metal whose hydroxo complexes are able to undergo olation and / or oxolation in aqueous solution.

[0022] Item 9: the method of any one of items 1 to 8, wherein the metal comprises a transition metal of group 6 to 10, for example comprises Fe.

[0023] Item 10: the method of item 9, wherein the metal oxide comprises FesO4.

[0024] Item 1 1 : the method of any one of items 1 to 10, wherein the nanoparticles B are obtained by a solvothermal process, wherein heat is applied to a precursor of the metal dissolved in a solvent, for example the metal precursor is Fe(acac)3.

[0025] Item 12: the method of item 1 1 , wherein the solvent is an alcohol, for example benzyl alcohol.

[0026] Item 13: the method of item 1 1 or 12, wherein the metal precursor is heated at a temperature of 175°C to 350°C.

[0027] Item 14: the method of any one of items 1 to 13, wherein, during step h), the amount of nanoparticles B in the aqueous mix, which is dispersed at step g), is such that the metal atoms in the metal oxide are in a concentration of from 0.5 mmol / L to 5 mol / L, for example of 0.5 to 500 mmol / L, for example of 15 to 150 mmol I L.

[0028] Item 15: the method of any one of items 1 to 14, wherein the amount of nanoparticles B in the aqueous mix to which the base is added at step e) is such that the metal atoms in the metal oxide are in a concentration of from 0.6 mmol / L to 6 mol / L, for example of 0.6 to 600 mmol / L, for example of 16 to 200 mmol I L.

[0029] Item 16: the method of any one of items 1 to 15, wherein the polysaccharide consists of only one polysaccharide.

[0030] Item 17: the method of any one of items 1 to 15, wherein the polysaccharide consists of a mixture of polysaccharides. Item 18: the method of item 17, wherein the mixture of polysaccharides comprises a polyanionic polysaccharide.

[0031] Item 19: the method of item 18, wherein the polyanionic polysaccharide comprises a repeating unit comprising a carboxylate group.

[0032] Item 20: the method of item 19, wherein the polyanionic polysaccharide is carboxymethyl -dextran.

[0033] Item 21 : the method of any one of the items 18 to 20, wherein the polyanionic polysaccharide represents 3% to 50%, preferably 7% to 27% of the total mass of the polysaccharide in the aqueous mix.

[0034] Item 22: the method of any one of items 1 to 17, wherein the polysaccharide is a non-polyanionic polysaccharide.

[0035] Item 23: the method of item 22, wherein the aqueous mix is devoid of a polyanionic polysaccharide.

[0036] Item 24: the method of any one of items 1 to 23, wherein the polysaccharide comprises dextran.

[0037] Item 25: the method of item 24, wherein the dextran has a weight average molar mass of 5000 to 200000 g / mol, for example of 10000 to 80000 g / mol, for example of 35000 to 45000 g / mol.

[0038] Item 26: the method of one of items 1 to 25, wherein the concentration of the polysaccharide in the aqueous mix, which is dispersed at step g), is of 20 to 1000 g / L, for example of 50 to 500 g / L.

[0039] Item 27: the method of one of items 1 to 26, wherein the concentration of the polysaccharide in the aqueous mix to which the base is added at step e) is of 25 to 1250 g / L, for example of 60 to 600 g / L.

[0040] Item 28: the method of one of items 1 to 27, wherein the amount of the polysaccharide and the amount of nanoparticles B in the aqueous mix at the end of step e) are such that the quantity of metal atoms in the metal oxide is of 0.5 mmol / 100 g of the polysaccharide to 500 mmol / 100 g of the polysaccharide, for example of 0.5 mmol / 100 g of the polysaccharide to 200 mmol / 100 g of the polysaccharide, for example of 5 mmol / 100 g of the polysaccharide to 100 mmol / 100 g of the polysaccharide.

[0041] Item 29: the method of one of items 1 to 28, wherein no polysaccharide is added to the mix or to the emulsion outside of step d), where the polysaccharide added is the polysaccharide coming from the aqueous solution provided at step a).

[0042] Item 30: the method of one of items 1 to 29, wherein no nanoparticles B are added to the mix or to the emulsion outside of step d), where the nanoparticles added are the nanoparticles coming from the aqueous suspension provided at step b). Item 31 : the method of any one of items 1 to 30, wherein the surfactant is a non-ionic surfactant.

[0043] Item 32: the method of any one of items 1 to 31 , wherein the surfactant has a hydrophilic-lipophilic balance (HLB) of 2 to 8.

[0044] Item 33: the method of any one of items 1 to 32, wherein the surfactant comprises a nonionic surfactant comprising: as a hydrophilic group, a polyethyleneglycol or a polyglycerol chain, as a hydrophobic group, a polyester of a monohydroxylated fatty acid, for example the surfactant comprises polyglycerol polyricinoleate (PGPR).

[0045] Item 34: the method of item 33, wherein the surfactant consists of PGPR.

[0046] Item 35: the method of one of items 1 to 34, wherein the amount of surfactant in the mix during step g) represents 5 to 240%, for example 20 to 150% of the mass of the aqueous mix dispersed at step g).

[0047] Item 36: the method of any one of items 1 to 35, wherein the oil phase provided at step c) comprises the surfactant dissolved therein at a concentration of 0.5 to 20 wt%, for example of 1 to 10 wt% relative to the total weight of the oil phase.

[0048] Item 37: the method of one of items 1 to 36, wherein the base is a strong base in water.

[0049] Item 38: the method of item 37, wherein the base is NaOH.

[0050] Item 39: the method of any one of items 1 to 38, wherein the amount of base added at step e) is of 0.3 to 30 mmol of OH- 1 g, for example of 0.5 to 10 mmol of OH7g of polysaccharide in the aqueous mix at step e).

[0051] Item 40: the method of one of items 1 to 39, wherein, just after adding the base and before adding the cross-linking agent, the aqueous mix is kept at a temperature below 10°C, for example below 5°C, for 30 seconds to 15 minutes, for example for 2 to 7 minutes.

[0052] Item 41 : the method of item 40, wherein, just after adding the base and before adding the cross-linking agent, the aqueous mix is kept at a temperature below 5°C, for 2 to 7 minutes.

[0053] Item 42: the method of any one of items 1 to 41 , wherein the cross-linking agent is trisodium trimetaphosphate (STMP). Item 43: the method of one of items 1 to 42, wherein no cross-linking agent is added to the mix or to the emulsion outside of step f).

[0054] Item 44: the method of one of items 1 to 43, wherein the cross-linking agent is added at step f) in an amount of 5 to 100 g per 100 g of polysaccharide, for example of 10 to 50 g per 100 g of polysaccharide.

[0055] Item 45: the method of one of items 1 to 44, wherein no strong base or acid is added to the mix or to the emulsion after step e).

[0056] Item 46: the method of one of items 1 to 45, wherein, from step e) to the beginning of step h), the temperature of the aqueous mix is kept below 10°C and the temperature of the emulsion is kept below 25°C, and wherein cross-linking comprises maintaining the emulsion at a temperature of 30°C to 90°C, for at least 10 minutes.

[0057] Item 47: the method of item 46, wherein, from step e) to the beginning of step h), the temperature of the aqueous mix is kept below 5°C and the temperature of the emulsion is kept below 20°C, and wherein cross-linking comprises maintaining the emulsion at a temperature of 40°C to 80°C, for at least 15 minutes.

[0058] Item 48: the method of any one of items 1 to 47, wherein the aqueous mix dispersed at step g) comprises a concentration of a water-soluble monovalent salt of at least 1 mol / L, for example of 2 to 4 mol / L.

[0059] Item 49: the method of any one of items 1 to 47, wherein the aqueous solution provided at step a) is saturated with the monovalent salt.

[0060] Item 50: the method of item 48 or 49, wherein the monovalent salt is NaCI.

[0061] Item 51 : the method of any one of items 1 to 50, wherein the amount of the aqueous mix dispersed at step g) represents 2 to 20 vol%, for example 3 to 10 vol% of the final mixture.

[0062] Item 52: the method of any one of items 1 to 51 , wherein the solvent of the oil phase is a vegetable oil.

[0063] Item 53: the method of item 52, wherein the vegetable oil is sunflower oil.

[0064] Item 54: the method of any one of items 1 to 53, wherein recovering the particles A comprises separating the particles A from the oil phase by at least one centrifugation step, wherein after the centrifugation step, the supernatant is discarded, and the particles A are resuspended in an aqueous solution.

[0065] Item 55: the method of item 54, wherein the metal oxide is a magnetic oxide and wherein recovering the particles A further comprises, after the at least one centrifugation step, at least one step of magnetic separation, wherein the particles A are separated using a magnet from the aqueous solution in which they are suspended.

[0066] Item 56: the method of any one of items 1 to 55, wherein the aqueous suspension of the nanoparticles B further comprises gold nanoparticles.

[0067] Item 57: the method of item 56, wherein the gold nanoparticles are dextran-functionalized gold nanoparticles.

[0068] Item 58: particles obtainable by the method of any one of items 1 to 57.

[0069] Item 59: Particles A comprising a cross-linked polysaccharide matrix and metal oxide nanoparticles B, said particles A having a hydrodynamic size of 500 nm to 5 pm, a polydispersity index (PDI) of below 0.2, wherein the hydrodynamic size and the PDI are measured by dynamic light scattering on a suspension of the particles in pure water, and wherein the amount of the metal oxide in the particles A is such that the quantity of metal atoms in the metal oxide is of at least 0.2 mol / 100 g of particles A, for example of 0.2 to 1 .5 mol / 100 g of particles A, for example of 0.3 to 1 mol / 100 g of particles A.

[0070] Item 60: the particles A of item 59, having a hydrodynamic size of 700 nm to 3 pm wherein the hydrodynamic size is measured by dynamic light scattering on a suspension of the particles in pure water.

[0071] Item 61 : the particles A of item 59 or 60, further characterized in that they are spherical.

[0072] Item 62: the particles A of any one of items 59 to 61 , further characterized in that they have a Zeta potential of -20 to -35 mV for any pH in the range of 5 to 8.

[0073] Item 63: the particles A of any one of items 59 to 62, wherein the polysaccharide consists of only one polysaccharide.

[0074] Item 64: the particles A of any one of items 59 to 62, wherein the polysaccharide consists of a mixture of polysaccharides.

[0075] Item 65: the particles A of item 64, wherein the mixture of polysaccharides comprises a polyanionic polysaccharide.

[0076] Item 66: the particles A of item 65, wherein the polyanionic polysaccharide comprises a repeating unit comprising a carboxylate group. Item 67: the particles A of item 65 or 66, wherein the polyanionic polysaccharide is carboxymethyldextran.

[0077] Item 68: the particles A of any one of the items 65 to 67, wherein the polyanionic polysaccharide represents 3% to 50%, preferably 7% to 27% of the total mass of the polysaccharide.

[0078] Item 69: the particles A of any one of items 59 to 64, wherein the polysaccharide is a non-polyanionic polysaccharide.

[0079] Item 70: the particles A of any one of items 59 to 69, wherein the polysaccharide comprises dextran.

[0080] Item 71 : the particles A of item 70, wherein the dextran has a weight average molar mass of 5000 to 200000 g / mol, for example of 10000 to 80000 g / mol, for example of 35000 to 45000 g / mol.

[0081] Item 72: the particles A of any one of items 59 to 71 , wherein the polysaccharide is crosslinked via phosphodiester bonds.

[0082] Item 73: the particles A of any one of items 59 to 72, wherein the nanoparticles B have a size of between 1 .5 nm and 50 nm, for example of 5 nm to 25 nm, as measured by transmission electron microscopy (TEM).

[0083] Item 74: the particles A of any one of items 59 to 73, wherein the nanoparticles B are non-core-shell nanoparticles.

[0084] Item 75: the particles A of any one of items 59 to 74, wherein the nanoparticles B consist of the metal oxide.

[0085] Item 76: the particles A of any one of items 59 to 75, wherein the metal oxide is a magnetic oxide.

[0086] Item 77: the particles A of item 76, wherein the magnetic oxide is ferrimagnetic or ferromagnetic.

[0087] Item 78: the particles A of item 76 or 77, wherein the nanoparticles B have a magnetization per unit of mass of over 20 x 10-3A.m2 / gram.

[0088] Item 79: the particles A of any one of items 59 to 78, wherein the metal oxide is an oxide of a metal whose hydroxo complexes are able to undergo olation and oxolation in an aqueous solution.

[0089] Item 80: the particles A of any one of items 59 to 79, wherein the metal comprises a transition metal of group 6 to 10, for example comprises Fe. Item 81 : the particles A of item 80, wherein the metal oxide comprises FesO4.

[0090] Item 82: the particles A of any one of items 59 to 81 , comprising on average from 104to 106nanoparticles B, for example from 5.104to 5.105nanoparticles B.

[0091] Item 83: the particles A of any one of items 76 to 82, wherein the average magnetic moment of the particles A is of 10-15to 10-13A.m2, for example of 5.10-15to 5.1014A.m2.

[0092] Item 84: particles A of any one of items 59 to 83, wherein the particles A further comprise gold nanoparticles.

[0093] Item 85: the particles A of item 84, wherein the gold nanoparticles are dextran-functionalized gold nanoparticles.

[0094] Item 86: the particles A of any one of items 58 to 85 for use in a method of treatment of the human or animal body by therapy, preferably by photothermal therapy.

[0095] Item 87: the particles A of item 86 for use in the treatment of thrombosis.

[0096] Item 88: the particles A for use of item 87, wherein the method of treatment comprises contacting the thrombus with the particles A, preferably using a magnet, and heating the thrombus to a temperature of 50°C to 70°C, preferably of 57°C to 67°C, by irradiating the particles A in contact with the thrombus with a laser.

[0097] Item 89: a method of treatment of a thrombosis in a patient in need thereof comprising administering to the patient a therapeutically effective amount of the particles A of anyone of items 58 to 85.

[0098] Item 90: the method of treatment of item 89, wherein the method of treatment further comprises contacting the thrombus with the particles A, preferably using a magnet, and heating the thrombus to a temperature of 50°C to 70°C, preferably of 57°C to 67°C, by irradiating the particles A in contact with the thrombus with a laser.

[0099] Brief description of the drawings

[0100] Fig. 1 shows a TEM image of a particle A according to the present disclosure.

[0101] Fig. 2 shows the evolution over time of the size and polydispersity of a sample of particles A according to the present disclosure dispersed in water.

[0102] Fig. 3 shows the evolution over time of magnetic nanoparticles B (MNP) retention in the particles A (HP) ([Fe] = 0.01 M) in water. Fig. 4 shows the temperature elevation obtained after 5 min irradiation of a suspension of particles A (HP) according to the present disclosure at different iron concentrations, using a laser (808 nm) at different powers. Heating was recorded with an infrared camera.

[0103] Fig. 5 shows the temperature elevation obtained after 5 min irradiation of various suspensions of particle A (hybrid polysaccharide / FesC particles (HPs) vs hybrid polysaccharide / FesO gold particles (AuFeHPs)) according to the present disclosure at different iron concentrations, using a laser (808 nm) at different powers. Heating was recorded with an infrared camera.

[0104] Fig. 6a (left) shows the effect of different levels of hyperthermia (1 h at 37°C, 50°C, 60°C or 70°C) on in vitro clots. The hemoglobin (HB) release (cyanmethemoglobin dosage) was normalized to the clot weight.

[0105] Fig. 6b (right) shows the effect of different levels of hyperthermia (1 h at 50°C, 55°C, 60°C or 65°C) on in vitro clots. The hemoglobin (HB) release (cyanmethemoglobin dosage) was normalized to the clot weight.

[0106] Fig. 7 shows in vitro static physical thrombolysis with different treatment conditions: laser only (PBS) and HP + MT + laser. The hemoglobin (HB) release (cyanmethemoglobin dosage) was normalized to the clot weight. Laser: 15-min laser irradiation (808 nm, 2 W / cm2). HP: particles A. MT: 3-min magnetic targeting (magnet placed below the tube to attract HPs to the clot).

[0107] Fig. 8a (left) shows comparison of HP-mediated photothermal thrombolysis (HP + MT + Laser) with the standard thrombolytic agent (rt-PA) at different concentrations (15 or 150 pg / mL, respectively equivalent to 1 or 10 mg / kg). The release of hemoglobin (HB) is measured with cyanmethemoglobin dosage. HP: particles A. MT: 3-min magnetic targeting (magnet placed below the tube to attract HPs to the clot). Laser: 15-min laser irradiation (808 nm, 2 W / cm2).

[0108] Fig. 8b (right) shows comparison of HP-mediated photothermal thrombolysis (HP + MT + Laser) with the standard thrombolytic agent (rt-PA) at different concentrations (15 or 150 pg / mL, respectively equivalent to 1 or 10 mg / kg). The release of fibrin degradation products in supernatant is measured through fluorescence measurement (Aex = 650 nm, Aem = 668 nm). HP: particles A. MT: 3-min magnetic targeting (magnet placed below the tube to attract HPs to the clot). Laser: 15-min laser irradiation (808 nm, 2 W / cm2).

[0109] Fig. 9 shows the results of a magnetic targeting flow assay. Three different conditions were tested in both venous and arterial flow rates: no magnet during particles A according to the present disclosure (HP) flowing and NaCI rinsing (No magnet); magnet during HP flowing but no magnet during rinsing (Magnet HP); magnet during HP flowing and magnet during rinsing (Magnet HP + R). n = 3 different batches of fluorescent HPs.

[0110] Fig. 10 is in vivo fluorescence image showing hybrid particles (red) targeted to the clot (green) after magnetic targeting from both sides of the occluded vessel.

[0111] Fig. 1 1 shows hydrodynamic size (Z-average) of suspensions in water of particles A depending on the proportion of carboxymethyl dextran in the polysaccharide mix.

[0112] Detailed description

[0113] Metal oxide

[0114] As herein defined, a metal oxide can be an oxide of any metal, including alkali metals, alkaline earths, transition metal, post transition metals, metalloids, lanthanides, and actinides.

[0115] The nanoparticles B can comprise a mixture of particles of different metal oxides. In which case, the metal oxide is a mixture of metal oxides.

[0116] As defined herein, the metal oxide can be a mixed oxide of two or more metals.

[0117] As defined herein, a metal oxide particle is non-coated when its surface is predominantly the metal oxide surface. This includes the case where the particle’s surface comprises ligands that can exchange with the water molecules of the solvent.

[0118] As defined herein a metal oxide nanoparticle is a nanoparticle that comprises at least an external shell of the metal oxide.

[0119] It is presumed that the good metal oxide load and polydispersity of the particles A obtained by the presently disclosed process involves the controlled aggregation of the particles B via a mechanism involving the metal oxide surface.

[0120] Synthesis of the nanoparticles B

[0121] In the solvothermal process, a conventional oven or microwaves can be used for applying the heat.

[0122] The size of the nanoparticles B (largest dimension of the particle, i.e., in the case of a sphere, its diameter) can be measured by transmission electron microscopy (TEM) according to the following procedure: a drop of the suspension of nanoparticles is deposited on a carbon coated copper grid placed on a filter paper. Images are then recorded. The size is measured by analyzing the image with an image treatment software. Magnetic oxide

[0123] As defined herein, a magnetic oxide is an oxide that is being attracted to a magnet and / or able to become a permanent magnet. A magnetic oxide can be, for example, a paramagnetic oxide, a ferrimagnetic oxide or a ferromagnetic oxide.

[0124] An advantage of using ferrimagnetic or ferromagnetic nanoparticles B is that it allows to use a magnetic separation step to purify the particles. Furthermore, ferrimagnetic or ferromagnetic nanoparticles B are required when the particles A are destined to be used in a non-pharmaceutical treatment of thrombosis involving mechanical action on the thrombus via magnetic stimulation. In such cases, the metal oxide preferably also presents photothermal properties. FesO (for example magnetite) is both ferrimagnetic and photothermal.

[0125] Order of addition of the reactants

[0126] For the aggregation process to proceed as desired and to obtain aggregates of the appropriate size in the aqueous mix, the inventors have observed that it was necessary to mix the nanoparticles B and the polysaccharide before adding the base and that it was necessary to add the cross-linking agent after having added the base.

[0127] Role of the base

[0128] The base destabilizes the metal oxide nanoparticles B and causes their aggregation. However, with the presently disclosed method, this aggregation process remains controlled and helps obtaining particles A with good polydispersity and metal oxide loading.

[0129] It has been observed that adding the base to the polysaccharide before adding the nanoparticles leads to uncontrolled aggregation.

[0130] After the addition of the base, controlling the temperature of the mix and time it is left to stand can participate in the control of the aggregation of the nanoparticles B.

[0131] The base also facilitates the cross-linking of the polysaccharide.

[0132] Oil phase

[0133] The oil phase is a liquid hydrophobic and lipophilic phase.

[0134] Surfactant

[0135] As defined herein, the surfactant can be a mixture of surfactants.

[0136] When the HLB of the surfactant is of 2 to 8, it favors the w / o emulsion over o / w emulsions. Note that when mixtures of surfactants are used, the mixture can comprise surfactants having a HLB outside this range.

[0137] An example of a surfactant comprising a polyglycerol chain and a polyester of a monohydroxylated fatty acid is PGPR.

[0138] An example of a surfactant comprising a polyethyleneglycol chain and a polyester of a monohydroxylated fatty acid is PEG-30 dipolyhydroxystearate.

[0139] Polysaccharide

[0140] As herein defined, the polysaccharide can be a mixture of polysaccharides.

[0141] It should be understood that a polysaccharide is considered a polyanionic polysaccharide when it comprises monomer units (i.e. repeating units) which are anionic at neutral pH. Thus, carboxymethyldextran with an average of about 1 carboxymethyl group for every five glucose units is polyanionic, whereas TRITC-dextran, which contains an average of less than 1 TRITC for 100 glucose units is not polyanionic.

[0142] Even when the polysaccharide mix comprises a polyanionic polysaccharide it is possible to obtain satisfying polydispersities.

[0143] Introducing anionic groups in the particle, in particular carboxylate groups, provides possibilities for further functionalization of the particles A.

[0144] As used herein, a particle comprising a cross-linked polysaccharide matrix is a particle wherein the cross-linked polysaccharide is the substance in which the other constituents of the particles are fixed, buried, etc., in other words the cross-linked polysaccharide constitutes the backbone of the particle.

[0145] Particles comprising a cross-linked polysaccharide matrix, such as the particles A of the present disclosure, are often referred to as hydrogel particles.

[0146] The particles A of the present disclosure are hybrid particles in the sense that they comprise an organic matrix and metal oxide (and thus inorganic) nanoparticles.

[0147] Cross-linking agent

[0148] It has been observed that adding the cross-linking agent trisodium trimetaphosphate (STMP) to the suspension of nanoparticles B before mixing them with the polysaccharide solution leads to uncontrolled aggregation. After the addition of the cross-linking agent, the control of the temperature of the mix not only influences the aggregation of the nanoparticles but it is also useful to avoid unwanted cross-linking before the cross-linking step h).

[0149] Some crosslinking agents that may be used in the method of the present disclosure, such as STMP or POCI3, result in the formation of phosphodiester bond between the polysaccharide chains. A phosphodiester bond liking two groups R has the following formula:

[0150] R-O-P(=O)(-Oj-O-R.

[0151] Salt

[0152] Adding a salt to the aqueous phase helps stabilizing the emulsion formed and facilitates reactions between species of the same charge, if any, during the crosslinking step, by screening the charges. Such species can be, for example, the cross-linking agent, the polysaccharide activated by the base, partially crosslinked polysaccharide chains when the crosslinking results in conferring a charge to the polysaccharide. This is the case, for example, when STMP is used as the cross-linking agent.

[0153] As defined herein, a monovalent salt is an ion pair wherein the anion has a charge of -1 and the cation has a charge of +1 .

[0154] Characteristics of the particles A

[0155] The hydrodynamic size (also called Z-average size) and the PDI are defined by the ISO norms: ISO 22412:2017 and ISO 22412:2017 respectively.

[0156] The hydrodynamic size is measured in water by dynamic light scattering (DLS), for example using an instrument such as a Zetasizer Nano ZS (Malvern Instruments, Orsay, France).

[0157] The hydrodynamic size of the presently disclosed particles can depend on the salinity of the solution in which they are dispersed (for example, they can diminish in size when salt is added). The presently disclosed particles are stable in a saline solution, even when no surfactant is added to the suspension.

[0158] The Zeta potential is defined by ISO norms ISO 13099-2:2012 and ISO 13099-1 :2012.

[0159] The Zeta potential is measured by electrophoretic light scattering (ELS) in 1 mM KOI, for example, using a Zetasizer Nano ZS (Malvern Instruments, Orsay, France).

[0160] The average magnetic moment of particles A can be measured according to the following procedure. First the number of particles A per unit of volume is measured by counting them with a counting chamber (for example a Kova slide counting chamber) through optical microscopy. Then the saturation magnetization of a given volume of the suspension of particles A is measured with a vibrating-sample magnetometer (VSM). The average magnetic moment of particles A is obtained by dividing this magnetization by the number of particles A in the sample.

[0161] The average number of nanoparticles B per particle A can be measured, for example, by elemental analysis, or in the case of magnetic nanoparticles B by dividing the magnetic moment of the particles A by the magnetic moment of one nanoparticle B. The magnetic moment of a nanoparticle B can be computed knowing the magnetic moment per unit mass of the magnetic oxide of which the nanoparticle B is composed and knowing the size (and therefore the mass) of the nanoparticle B.

[0162] The particles A of the invention can be used, for example as part of a pharmaceutical composition, to treat a thrombus via photothermal therapy. In this case, it has been shown that the treatment was more efficient when the thrombus was heated at temperatures of 50 to 70°C and preferably of 57 to 67°C. The heating is conveniently obtained by directing the particles A of the invention to the thrombus using a magnet and irradiating them with a laser. Preferably the laser emits in a wavelength range of 650 nm to 1 mm, preferably of 750 to 900 nm. In order to control the heating temperature, an infrared camera can be used.

[0163] Photothermal therapy (PTT) refers to efforts to use electromagnetic radiation (most often in infrared wavelengths) for the treatment of various medical conditions, including cancer. In this approach a photosensitizer is excited with light. This activation brings the sensitizer to an excited state from where it can release vibrational energy and heat surrounding materials and molecules, which is used to destroy a target, such as a cell, a thrombus, a virus etc. The particles A of the invention are efficient photosensitizers for photothermal therapy.

[0164] As used herein, the expression "therapeutically effective amount" as above described means a sufficient amount of the particle for the treatment of thrombosis. It will be understood, however, that this amount will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination with the specific agonist employed; and like factors well known in the medical arts.

[0165] Experiments

[0166] Materials

[0167] Dextran 40 kDa was provided by Pharmacosmos (Holbaek, Denmark). TRITC-Dextran 40 kDa was purchased from TDB Consultancy (Uppsala, Sweden). Iron (III) acetylacetonate (> 99.9%), Benzyl alcohol anhydrous (99.8%), Sodium trimetaphosphate (STMP), Sodium Dodecyl Sulfate (SDS) and Sucrose were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Polyglycerol polyricinoleate (PGPR) was obtained from Palsgaard France S.A.S. (Lyon, France). Sunflower oil (Lesieur - Huile Coeur de Tournesol, Lesieur S.A.S, Asnieres-sur-Seine, France) was purchased from a local supermarket (Monoprix, Paris, France).

[0168] Physico-chemical characterization

[0169] Particles were studied for particle morphology, size and zeta potential distributions, mass concentration, and composition.

[0170] Particle morphology was visualized by transmission electron microscopy (TEM) (Philips FEI Tecnai 12, Amsterdam, Netherlands) at the ImagoSeine core facility of the Institut Jacques Monod. The particles A (hereafter also referred to as hybrid particles) were negatively stained with 1 % (w / v) uranyl acetate for 30s. Images were obtained by depositing a drop of particles suspension on carbon coated copper grids placed on a filter paper (in the case of the nanoparticles B) or on formvar coated copper grids placed on a filter paper (in the case of the particles A).

[0171] Hydrodynamic size and Zeta potential (^-potential) were measured by dynamic light scattering (DLS) and electrophoretic light scattering (ELS) respectively (Zetasizer Nano ZS, Malvern Instruments, Orsay, France). Samples were dispersed in distilled water or saline for size and in 1 mM KCI for ^-potential determination. All runs were performed at 25°C in triplicate.

[0172] Mass concentration was determined by freeze-drying a sample of the hybrid particles to determine their total mass and the mass of iron in the hybrid particles was determined by iron dosage (see below).

[0173] Particle composition was analyzed through Fourier Transformed Infrared Spectroscopy (FTIR) with a Bruker Tensor 27 FTIR. Samples were analyzed under the form of KBr pellets.

[0174] Iron dosage: The iron concentration of the particles (hybrid particles as well as metal oxide nanoparticles) was determined through a colorimetric quantification: iron in samples was destroyed with HNO3 at 7 M. H2O2 was added to oxidize the iron ions. The acidic destruction is performed by heating at 70-75°C for 7 hours. Then, iron concentration was determined by thiocyanate colorimetry: the samples were diluted with water and then potassium thiocyanate was added. The absorption was measured at 478 nm with a Varioskan LUX multiplate reader (Thermo Fisher Scientific, Massachusetts, United States). The corresponding iron concentration was found with standard curves obtained with iron standard at different concentrations.

[0175] Average magnetic moment: the average magnetic moment of the hybrid particles was measured according to the following procedure. First the number of particles A per unit of volume is measured by counting them with a Kova slide counting chamber through optical microscopy. Then the saturation magnetization of a given volume of the suspension of particles A is measured with a vibrating-sample magnetometer (VSM). The average magnetic moment is obtained by dividing this magnetization by the number of hybrid particles in the sample.

[0176] Preparation of nanoparticles (NP or MNP, corresponding to nanoparticles B)

[0177] Synthesis: All the synthesis was carried out in a glovebox (O2 and H2O < 0.5 ppm). 400 mg of iron (III) acetylacetonate were dissolved in 10 ml of benzyl alcohol. Then, nanoparticle synthesis was carried out in a microwave oven (Monowave 300 from Anton Paar). The temperature of the iron (III) acetylacetonate solution was increased up to 250°C in 20 minutes then maintained constant for 30 minutes to obtain the NPs.

[0178] Purification: The resulting suspension was successively washed with hydrochloric acid (10-2mol.L-1) and dichloromethane by sonication (30 s) and mechanical agitation (1 h). Each time, the aqueous phase comprising the NPs was recovered after centrifugation.

[0179] The remaining dichloromethane and benzyl alcohol in the aqueous phase were evaporated overnight and removed by different steps of ultrafiltration in Amicon® Ultra centrifugal filters (100 kDa). The obtained NP suspension had an iron content of 0.32 mol / L.

[0180] Size of the NPs: the size of the NPs was measured by analyzing the images of the NPs obtained by TEM with the Fiji software. A size of 9 nm with a standard deviation of 2-3 nm was measured.

[0181] Preparation of microwave-assisted dextran-functionalized gold nanoparticles (AuNPs)

[0182] After dissolving 38.8 mg of gold chloride III trihydrate (solution A) and 59.1 mg of Dextran 40 kDa (solution B) in 5 mL of water each, 250 pL of solution A and 500 pL of solution B are dispersed in 19 ml of water. The pH is then adjusted to 1 1 with a 1 M NaOH solution. Dextran-functionalized gold nanoparticle (AuNP) synthesis was carried out in a Monowave 300 from Anton Paar at 100°C for 10 minutes. The suspension is then concentrated 100-fold by ultrafiltration through Amicon® Ultra centrifugal filters (100 kDa).

[0183] Preparation of hybrid polysaccharide / FesCT particles (HP, corresponding to particles A)

[0184] Synthesis: HPs were obtained via a water-in-oil (w / o) emulsification combined with a crosslinking process of the aqueous phase.

[0185] First, a polysaccharide solution comprising 3 g dextran 40 kDa, 3.5 g NaCI, and 10 g H2O was prepared. For fluorescent HPs, 5% of the dextran mass was replaced by TRITC-Dextran 40 kDa. STMP was dissolved in water to obtain a 30% w / v STMP solution. Then, the organic phase made of 27.6 g of sunflower oil and 7% w / w PG PR was prepared and cooled down for 20 min at -20°C.

[0186] In the meantime, the aqueous phase made of 1704 mg of the polysaccharide solution was introduced in a 5 mL eppendorf tube and placed in an iced bath to limit the cross-linking process, which could occur when STMP is added. 310 pL of the NPs suspension (0.32 M Fe) were added in the aqueous phase and vortexed for 20 s. Then, 170 pL of 10 M NaOH were added for polysaccharide activation and homogenized in the aqueous phase with a vortex for 2 min. The aqueous phase is left in the ice bath for 2 min before being further vortexed for 1 min. 341 pL of the STMP solution was then added into the aqueous phase to cross-link polysaccharide chains after emulsification. The aqueous phase was homogenized with a vortex for 20 s. Next, emulsification was achieved by the dropwise addition of 1 .9 mL of the aqueous phase into the organic phase and dispersed with a stand-disperser (Polytron PT 3100 D, dispersing aggregate PT-DA 07 / 2 EC-B101 , Kinematica, Luzernerstrasse, Switzerland) at 30,000 rpm for 6 min in an ice bath. The obtained w / o emulsion was placed in an oven at 50 °C for 20 min to promote crosslinking of the polysaccharide and obtain the HPs.

[0187] When NPs are introduced in the aqueous mix after the base, NPs aggregate in an uncontrolled manner. The aggregates are visible to the naked eye in the aqueous mix and sediment.

[0188] When STMP is dissolved in the NPs suspension to bring the NPs as late as possible (to avoid NPs aggregation in contact to the base), the same is observed. The NPs aggregate an uncontrolled manner in presence of the STMP alone. The HPs obtained were larger than 5 pm and had a PDI much higher than 0.2.

[0189] Purification: he crosslinked emulsion was homogenized and split into 4 ultracentrifugation tubes. 1 mL of PBS 1 X was added in each tube. The 4 tubes were ultracentrifuged (Optima MAX-XP, Ultracentrifuge, Beckman Coulter, Brea, California, United States) for 1 h30 at 6,000 g. The oily phase of the supernatant was removed and 5 mL of PBS 1 X were added in each tube. The tubes were vortexed and then sonicated in ice bath for 30 min for pellet redispersion. The tubes were vortexed again to finish pellet redispersion and the content of the 4 ultracentrifugation tubes was transferred to a Falcon® 50 mL. Then, the mixture was magnetically separated. The magnetic part was collected and redispersed in 5 mL of 0.04% Sodium Dodecyl Sulfate (SDS) solution. The mixture was then vortexed until total redispersion of the magnetic part and transferred to a clean tube. This magnetic separation process was performed one more time in 5 mL of 0.04% SDS and then one time in 5mL of ultrapure water to purify the hybrid particles (HPs). Then, the mixture was filtrated with a 30-pm membrane to remove oil residues and a few aggregates of hybrid particles. A 5-pm filtration step was added to be safe for further animal experiments. The resulting suspension was magnetically separated one last time in 5 mL of ultrapure water. Finally, HPs were stored in water at 4°C. Stable freeze-dried HPs could be obtained with the LyoVac GT2 freeze dryer (SRK Systemtechnik, Riedstadt, Allemagne) after -80°C freezing for 1 h in a 1 % sucrose solution.

[0190] In water, the hydrodynamic size of the HPs was 1 .5 pm and their polydispersity was of about 0.15. In a 0.9 wt% solution of NaCI in water, their hydrodynamic size was 0.9 pm and their polydispersity was 0.1 1 . The Zeta potential of the HPs was -26 mV. Their average magnetic moment was of 10-14A.m2per HP and the number of NPs per HP was 105.

[0191] The mass of iron in the HPs was of 28% of the total mass of the HPs (0.5 mol of Fe per 100 g of HPs). Synthesis of hybrid polvsaccharide / maqnetite / qold particles (AuFeHPs, corresponding to particles A) AuFeHPs were synthetized following the same protocol as hybrid polysaccharide / magnetite particles (HPs) synthesis with a few adjustments: instead of adding 310 pL of magnetite nanoparticles (0.3 M Fe), 155 pL of magnetite nanoparticles (0.3 M Fe) and 155 pL of dextran-functionalized gold nanoparticles (100-fold concentrated) were added. During the purification step, PBS 1 X was replaced by ultrapure water.

[0192] The size of AuFeHPs is 950 nm in PBS with a PDI of 0.05. The surface charge of AuFeHPs is -28 mV.

[0193] Synthesis of hybrid carboxymethyl dextran-modified polysaccharide / magnetite particles (CMD-HPs, corresponding to particles A)

[0194] CMD-HPs were synthetized following the same protocol as hybrid polysaccharide / magnetite particles (HPs) synthesis by replacing 5%, 10%, 25% or 50% of total dextran mass by carboxymethyl-dextran 40 kDa (CMD).

[0195] Photothermal assay:

[0196] Temperature elevation was measured after 5 min irradiation of a suspension of HPs or AuFeHPs at different iron concentrations, using a laser (808 nm) at different powers. Heating was recorded with an infrared camera.

[0197] Platelet binding assay:

[0198] To investigate the capacity of hybrid particles to target the thrombus magnetically, we assessed if they could be retained by a magnet in flow conditions on activated platelets. To do this, the microchannels of a Vena8 Fluoro+biochip (Cellix Ltd, Dublin, Ireland) were coated with a fibrillar type I collagen solution at 200 pg / ml (equine tendon collagen, Collagen Reagens HORM®, Takeda) overnight at 4°C and rinsed with NaCI 0.9% before use. Human whole blood (EFS, Bichat Hospital, Paris, France), collected in the PPACK tubes and labeled with 2 pM DIOC6 (3,3'-Dihexyloxacarbocyanine) (Life Technologies SAS, Saint-Aubin, France), was perfused at arterial shear stress for 5 min to induce platelet activation and aggregation. Platelet aggregation through contact with collagen was visualized in real-time with phasecontrast microscopy (Axio Observer, Carl Zeiss Microscopy, Oberkochen, Germany). After rinsing with NaCI 0.9%, fluorescent hybrid particles (HPs) (0.9 mM Fe) were injected into the channels for 5 min at venous (4 pL / min) or arterial (60 pL / min) flow rates. During this injection, a magnet was placed above the channel or not. Their accumulation onto activated platelet aggregates was monitored in real-time. Channels were then washed for 5 min (arterial condition) or 10 min (venous condition) with NaCI 0.9%. During this step, the magnet was kept above the channel or not. Finally, the area percentage covered by the fluorescent HPs in the region of interest (ROI) on each channel was analyzed with Fiji. Intensity settings were kept the same for all replicates. Three different conditions were tested in both venous and arterial flow rates: no magnet during HP flowing and NaCI rinsing (No magnet); magnet during HP flowing but no magnet during rinsing (Magnet HP); magnet during HP flowing and magnet during rinsing (Magnet HP + R). n = 3 different batches of fluorescent HPs. MNP retention in HPs:

[0199] At each time point, the HP suspension ([Fe] = 0.01 M) is centrifuged at 3,000 g for 5 min in order to obtain a pellet of HPs while keeping MNPs released from HPs in suspension. The quantity of MNPs in the supernatant was determined by iron dosage. The percentage of MNPs retained in HPs was computed as followed: 100 -

[0200] Effect of hyperthermia on in vitro clots:

[0201] Human whole blood was collected in sodium citrate tubes, and 200 pL of blood were mixed with CaCIz (10 mM) and thrombin (0.1 UI / mL) in glass tubes. The tubes were placed in a bath at 37°C for 1 h. The resulting clots were washed four times in PBS, weighed, transferred in clean glass tubes containing PBS, then washed again four times. Supernatant was sampled for further dosage of released hemoglobin. The clots were then placed in baths at different temperatures (37°C, 50°C, 55°C, 60°C, 65°C, 70°C) for 1 h. Supernatant was sampled again for further dosage of released hemoglobin. The supernatants were frozen at -20°C for 24 h. Quantification of released hemoglobin (HB) was performed by thawing the supernatants, centrifugating them (collecting supernatants) and then mixing 20 volumes of Drabkin’s reagent (200 mg / L potassium ferricyanide, 50 mg / L potassium cyanide, 140 mg / L potassium dihydrogen phosphate, 1 % (v / v) Tween 20, pH = 7.0-7.4) with 1 volume of supernatant (cyanmethemoglobin dosage). After 5 min of incubation, absorbance is read at 540 nm with a Varioskan LUX multiplate reader.

[0202] In vitro static physical thrombolysis:

[0203] Human whole blood was collected in sodium citrate tubes, and 50 pL of blood were mixed with CaCIz (10 mM) and thrombin (1 UI / mL) in 1 .5 mL Eppendorf® tubes. The tubes were incubated at 37°C for 24h to obtain “old” clots. The resulting clots were washed three times in PBS, weighed, and transferred in clean glass tubes containing PBS. Supernatant was sampled for further dosage of released hemoglobin. PBS or hybrid particles were then added ([Fe] = 5 mM). The different treatment conditions were then performed: laser only (PBS); HP + MT + laser. Laser: 15-min laser irradiation (808 nm, 2 W / cm2). HP: hybrid particles. MT : 3-min magnetic targeting (magnet placed below the tube to attract HPs to the clot). After treatment, supernatant was sampled again for further dosage of released hemoglobin. The supernatants were frozen at -20°C for 24 h. Quantification of released hemoglobin (HB) was performed by thawing the supernatants, centrifugating them (collecting supernatants) and then mixing 20 volumes of Drabkin’s reagent (200 mg / L potassium ferricyanide, 50 mg / L potassium cyanide, 140 mg / L potassium dihydrogen phosphate, 1 % (v / v) Tween 20, pH = 7.0-7.4) with 1 volume of supernatant (cyanmethemoglobin dosage). After 5 min incubation, absorbance is read at 540 nm with a Varioskan LUX multiplate reader.

[0204] Comparison of HP-mediated photothermal thrombolysis with the standard thrombolytic agent:

[0205] To compare the physical thrombolysis with photo- and magneto-stimulated hybrid particles to the gold standard in thrombolytic therapy (rt-PA), we adapted the protocol of in vitro static physical thrombolysis. Human whole blood was collected in sodium citrate tubes and mixed with Alexa Fluor 647 fibrinogen conjugate (A647-Fg) from human plasma (75 pg / mL) to obtain later fluorescent clots and measure the release of fibrin degradation products after the different treatments. 50 pL of A647-Fg-blood were mixed with CaCIz (10 mM) and thrombin (1 UI / mL) in 1 .5 mL eppendorf tubes. The tubes were placed in a bath at 37°C for 1 h. The resulting A647-Fg-clots were washed four times in PBS and transferred in clean glass tubes (for physical treatment) or in a well-plate (for rt-PA treatment) containing PBS. Supernatant was sampled for further dosage of released hemoglobin and fluorescence detection. For clots treated physically, HPs ([Fe] = 2 mM) were added and only one treatment condition was performed: HP + MT + laser. HPs: hybrid particles. MT: 3-min magnetic targeting (magnet placed below the tube to attract HPs to the clot). Laser: 15-min laser irradiation (808 nm, 2 W / cm2). For rt-PA treatment, clots were incubated at 37°C with plasminogen (0.5 UI / mL) and rt-PA (15 or 150 pg / mL, respectively equivalent to 1 or 10 mg / kg) for 1 h with orbital agitation. After treatment, supernatant was sampled again for further dosage of released hemoglobin and fluorescence detection (HPs were removed from supernatant through magnetic separation). Fluorescence intensity (Aex = 650 nm, Aem= 668 nm) was measured in supernatants with a Varioskan LUX multiplate reader. The supernatants were then frozen at -20°C for 24 h. Quantification of released hemoglobin (HB) was performed by thawing the supernatants and mixing 20 volumes of Drabkin’s reagent (200 mg / L potassium ferricyanide, 50 mg / L potassium cyanide, 140 mg / L potassium dihydrogen phosphate, 1 % (v / v) Tween 20, pH = 7.0-7.4) with 1 volume of supernatant (cyanmethemoglobin dosage). After 5 min incubation, absorbance is read at 540 nm with a Varioskan LUX multiplate reader.

[0206] In vivo magnetic targeting:

[0207] Animal studies were performed on 6-8 weeks old C57BL6J mice (Janvier Labs, Le Genest Saint Isle, France). All experiments were adapted according to French (Decree 87 / 848) and European (2010 / 63 / EU) ethical guidelines. After an acclimatation period of one week in an enriched environment, mice received analgesia (subcutaneous injection of buprenorphine 0.05 mg / kg), then were anesthetized for the whole procedure (4% isoflurane inhalation for initiation then continuous 2% isoflurane inhalation). A midline abdominal incision was performed to expose the mesentery, which was gently laid out over a transparent Petri dish and placed under an intravital macroscope: Leica Z16 APO macroscope (Leica, Nanterre, France) equipped with an Orca Flash 4.0 LT camera (Hamamatsu, Hamamatsu City, Japan). A retro-orbital injection of 30 pL DIOC6 50 pM was performed to label leukocytes and platelets. A 1 mm large Whatmann chromatography paper band, previously soaked in 10 wt.% FeCIs was deposed on a mesentery vein for one minute before removal. The thrombus formation was observed in real time for 10 to 20 min by fluorescence macroscopy. Once the thrombus was formed, TRITC-labeled HPs were administered with a retro orbital injection (150 pL injection, [Fe] = 20 mg / kg). 15-20 minutes later, a magnet was placed close to the occluded vessel to retain HPs. 10-15 minutes later, the magnet was placed on the other side of the vessel to target both sides of the clot. An image was then acquired. At the end of the image acquisition, the animals were sacrificed. Fig. 1 is a TEM image of a HP colored with uranyl acetate. The aggregated NPs can be seen in black and the cross-linked polysaccharide matrix can be seen as the blurred round halo around the aggregated NPs.

[0208] Fig. 2 shows that the size and polydispersity of the HPs remain stable in water for over 3 months.

[0209] Fig. 3 shows the stability of the HPs. In particular, it shows that the MNPs are retained in the HPs for over 90 days.

[0210] Fig. 4 shows that the HPs have a photothermal response that can be controlled precisely with the iron concentration and the laser power. There is a wide range of temperature elevation, and the temperatures reached are largely enough for a thrombolytic application.

[0211] Fig. 5 shows that the photothermal response of AuFeHP is higher than the photothermal response of HP at the same iron concentration.

[0212] Fig. 6a and Fig. 6b show the hyperthermia effect on in vitro clots is maximal at 60°C.

[0213] Fig. 7 shows that the presence of the HP is required to obtain photothermal thrombolysis.

[0214] Fig. 8a and Fig. 8b (right) show that the method of the photothermal treatment of a thrombus according to the invention gives results that are comparable to the use of rtPA and could be even better. The hemoglobin released is higher and even if the fluorescence measured to assess the fibrin release is lower, it could be caused by deactivation of the fluorescence due to the heating and not by lower levels of fibrin release.

[0215] Fig. 7 and 8 also show that in order to obtain thrombolysis with the particles A of the invention a magnetomechanical stimulation of the particles is not needed.

[0216] Fig. 9 shows that HPs are almost not retained on activated platelets without magnet. In the presence of a magnet, HPs are stopped despite the venous or arterial flow, whether the magnet is kept during the rinsing step or not. This is a proof of concept for the magnetic targeting of the thrombus.

[0217] Fig. 10 shows that, in the presence of a magnet, TRITC-labeled HPs can target the clot from both sides of the occluded vessel.

[0218] Fig. 1 1 shows the measured size of various samples of CMD-HPs. The polydispersity measured on these samples are presented in table 1 and show that the PDI depends on the proportion of CMD in the polysaccharide mix.

[0219] Table 1

Claims

CLAIMS1 . A method for the preparation of particles A, said particles A comprising a cross-linked polysaccharide matrix and metal oxide nanoparticles B, said method comprising the following steps: a) providing an aqueous solution comprising the polysaccharide; b) providing an aqueous suspension of the nanoparticles B; c) providing an oil phase; d) mixing said aqueous solution and said aqueous suspension to obtain an aqueous mix; e) adding a base to the aqueous mix; f) adding a cross-linking agent to the aqueous mix; g) dispersing the aqueous mix in the oil phase in presence of a surfactant to obtain a water / oil (w / o) emulsion; h) crosslinking the polysaccharide to obtain the particles A, wherein the crosslinking comprises the reaction between a crosslinking agent and the polysaccharide to obtain the cross-linked polysaccharide matrix comprised in the particles A; i) recovering the particles A; wherein steps d) to i) are performed successively in this order and wherein the nanoparticles B introduced at step d) are non-coated metal oxide nanoparticles.

2. The method of claim 1 , wherein the surfactant comprises a nonionic surfactant comprising: as a hydrophilic group, a polyethyleneglycol or a polyglycerol chain, as a hydrophobic group, a polyester of a monohydroxylated fatty acid, for example, the surfactant comprises polyglycerol polyricinoleate (PGPR).

3. The method of claim 1 or 2, wherein the base is a strong base in water and wherein the amount of base added at step e) is of 0.3 to 30 mmol of OH- 1 g, for example of 0.5 to 10 mmol of OFT / g of polysaccharide in the aqueous mix at step e).

4. The method of any one of claims 1 to 3, wherein the metal of the metal oxide comprises a transition metal of group 6 to 10, for example comprises Fe.

5. The method of claim 4, wherein the metal oxide comprises FesO4.

6. The method of any one of claims 1 to 5, wherein the amount of the polysaccharide and the amount of nanoparticles B in the aqueous mix at the end of step e) are such that the quantity of metal atoms in the metal oxide is of 0.5 mmol / 100 g of the polysaccharide to 500 mmol / 100 g of the polysaccharide, for example of 0.5 mmol / 100 g of the polysaccharide to 200 mmol / 100 g of the polysaccharide, for example of 5 mmol / 100 g of the polysaccharide to 100 mmol / 100 g of the polysaccharide.

7. The method of any one of claims 1 to 6, wherein, just after adding the base and before adding the cross-linking agent, the aqueous mix is kept at a temperature below 10°C, for example below 5°C, for 30 seconds to 15 minutes, for example for 2 to 7 minutes.

8. The method of any one of claims 1 to 7, wherein the amount of the aqueous mix dispersed at step g) represents 2 to 20 vol%, for example 3 to 10 vol% of the final mixture.

9. The method of any one of claims 1 to 8, wherein the metal oxide is a ferrimagnetic or ferromagnetic oxide and wherein recovering the particles A comprises separating the particles A from the oil phase by at least one centrifugation step, wherein after the centrifugation step, the supernatant is discarded, and the particles A are resuspended in an aqueous solution, and after the at least one centrifugation step, at least one step of magnetic separation, wherein the particles A are separated using a magnet from the aqueous solution in which they are suspended.

10. Particles obtainable by the method of any one of claims 1 to 9.1 1 . Particles A comprising a cross-linked polysaccharide matrix and metal oxide nanoparticles B, said particles A having a hydrodynamic size of 500 nm to 5 pm, a polydispersity index (PDI) of below 0.2, wherein the hydrodynamic size and the PDI are measured by dynamic light scattering on a suspension of the particles in pure water, and wherein the amount of the metal oxide in the particles A is such that the quantity of metal atoms in the metal oxide is of at least 0.2 mol / 100 g of particles A, for example of 0.2 to 1 .5 mol / 100 g of particles A, for example of 0.3 to 1 mol / 100 g of particles A.

12. The particles A of claim 1 1 , wherein the nanoparticles B have a size of between 1 .5 nm and 50 nm, as measured by TEM.

13. The particles A of claim 1 1 or 12, wherein the metal oxide comprises FesCXi.

14. The particles A of any one of claims 1 1 to 13, wherein the average magnetic moment of the particles A is of 10-15to 10-13A.m2, for example of 5.10-15to 5.1014A.m2.

15. The particles A of any one of claims 1 1 to 14, wherein the polysaccharide is crosslinked via phosphodiester bonds.

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