Crosslinked polysaccharide nanoparticles

Abstract

Crosslinked polysaccharide nanoparticles The present invention relates to cross-linked polysaccharide nanoparticles, which can be used as drug delivery systems. The herein disclosed cross-linked polysaccharide nanoparticles are obtained via an emulsion cross-linking process. The careful choice of the surfactants used for forming the emulsion allows to reliably obtain small particles with a low polydispersity. Moreover, these particles are stable in saline solutions. The cross-linked polysaccharide nanoparticles can be loaded with proteinaceous fibrinolytic or thrombolytic agents and could be used as drug delivery systems for the treatment of thrombosis.

Description

[0001] CROSSLINKED POLYSACCHARIDE NANOPARTICLES

[0002] Technical domain

[0003] The present disclosure relates to crosslinked polysaccharide nanoparticles obtained via an emulsion.

[0004] Background

[0005] Cardiovascular diseases are held responsible for a third of all deaths worldwide, leading with heart attacks and strokes, among which the latter is responsible for over 12.2 million cases each year. Notably, around 80 % of strokes are of ischemic nature, and the recommended care for this pathological blood clot (thrombus) is the quick systemic injection of a fibrinolytic drug, rtPA, which suffers from severe side effects, offers a short therapeutic window, and does not guarantee recanalization. It was also shown that the more complex nature of thrombi impedes their lysis.

[0006] It is therefore an object of the present disclosure to provide a biocompatible nanosystem targeting the thrombus, to deliver drugs for improved blood clot bursting and improved recovery.

[0007] Document WO2021249974A1 discloses cross-linked polysaccharide particles that may be functionalized with fucoidan and loaded with rtPA.

[0008] The particles described in WO2021249974A1 are biocompatible particles, prepared by the inverse emulsion / crosslinking method. When these particles were functionalized with fucoidan, a polysaccharide, which was demonstrated to have high affinity to P-selectin, a marker of activated platelets and endothelial cells, they could be used for thrombus targeting. It was shown in WO2021249974A1 , that rtPA could be loaded in the particles, and its amidolytic and fibrinolytic activities were maintained in vitro and in vivo. Thrombus targeting potential of these particles was validated in microfluidic assay under arterial and venous blood shear rates on recombinant P-selectin and activated platelet aggregates. The thrombolytic efficacy of the particles was tested in a murine model of acute ischemic stroke, revealing faster Middle Cerebral Artery recanalization and reduction in the brain infarct volume and blood-brain barrier permeability post-stroke, evidenced by laser speckle contrast imaging and MRI. Collectively, this proof-of-concept study demonstrated the potential of these particles for the precise treatment of acute thrombotic events.

[0009] However, the particles of WO2021249974A1 had several drawbacks. The size of the particles of WO2021249974A1 varied noticeably depending on the salinity of the dispersion medium and the particles were stable in a saline solution only if a surfactant was added to the saline solution. In WO2021249974A1 the sizes disclosed corresponds to measurements made in a saline solution in the presence of a surfactant. Particles sizes were higher when measured in pure water or in a saline solution devoid of a surfactant (in this latter case, moreover, the particles were unstable). It was in fact not possible to obtain particles with an average size below 500 nm as measured in pure water or in a saline solution devoid of surfactant.

[0010] For therapeutic applications, however, smaller particles are advantageous as they allow better targeting and drug delivery (because of the higher surface over volume ratio). Due to margination of blood-borne objects in blood vessels, particles with size below 500 nm are also desired. Moreover, it is preferable that the particles are stable in saline conditions and in the absence of a surfactant.

[0011] Aiming to provide improved cross-linked polysaccharide particles that may be used as a drug delivery system, in particular for thrombus targeting and thrombolysis, the present disclosure provides novel particles with a smaller size. Moreover, it has been observed that the size of the particles does not vary depending on the ionic strength of the aqueous solution in which they are dispersed. Moreover, the particles are reliably obtained with a low polydispersity.

[0012] Summary

[0013] The method of the invention relies on the selection of a particular mix of surfactants that allows the formation of a suitable emulsion, that in its turn yields particles comprising a cross-linked polysaccharide matrix with a smaller size than in the prior art. Moreover, it has been observed that the size of the particles does not vary depending on the ionic strength of the aqueous solution in which they are dispersed and that the particles are stable in a saline aqueous solution even in the absence of added surfactant. Moreover, the particles are reliably obtained with a low polydispersity.

[0014] As such the present invention relates to the following item:

[0015] Item 0: Method for the preparation of particles comprising a cross-linked polysaccharide matrix, said method comprising the following steps: a) providing an aqueous phase comprising the polysaccharide and a salt; b) providing an oil phase comprising an oil as a solvent; c) mixing the aqueous phase and the oil phase in presence of a mixture of a surfactant A and a surfactant B so as to obtain a water / oil (w / o) emulsion; d) crosslinking the polysaccharide comprised in the dispersed aqueous phase to obtain the particles, wherein the crosslinking comprises the reaction between a crosslinking agent and the polysaccharide to obtain the polysaccharide matrix; e) recovering the particles; wherein the surfactant A is 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, surfactant A is polyglycerol polyricinoleate; and wherein the surfactant B has a negative Cc value, for example, surfactant B is a nonionic surfactant having a negative Cc value, for example surfactant B is a nonionic ethoxylated surfactant having a negative Cc value, for example surfactant B is a polysorbate having a negative Cc value, for example surfactant B is polyoxyethylene (20) sorbitan monooleate.

[0016] Item 1 : Method for the preparation of particles comprising a cross-linked polysaccharide matrix, said method comprising the following steps: a) providing an aqueous phase comprising the polysaccharide and a salt; b) providing an oil phase comprising an oil as a solvent; c) mixing the aqueous phase and the oil phase in presence of a surfactant so as to obtain a water / oil (w / o) emulsion; d) crosslinking the polysaccharide comprised in the dispersed aqueous phase to obtain the particles, wherein the crosslinking comprises the reaction between a crosslinking agent and the polysaccharide to obtain the polysaccharide matrix; e) recovering the particles; wherein the concentration of the salt in the aqueous phase, the oil, and the surfactant are selected so that the HLD at a temperature of 25°C is of 0 to 3, for example of 0 to 2.5, for example of 1 to 2.5.

[0017] Item 2: the method of item 0 or 1 , wherein step c) comprises stirring the oil phase using a rotor-stator homogenizer.

[0018] Item 3: the method of any one of items 0 to 2, wherein step c) comprises adding the aqueous phase dropwise to the oil phase, while stirring the oil phase, for example while stirring the oil phase using a rotor-stator homogenizer.

[0019] Item 4: the method of any one of items 0 to 3, wherein the surfactant is a non-ionic surfactant.

[0020] Item 5: the method of any one of items 1 to 4, wherein the surfactant is a mixture of at least two different surfactants.

[0021] Item 6: the method of item 5, wherein the mixture of surfactants comprises a surfactant A, which is 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 comprises polyglycerol polyricinoleate as the surfactant A.

[0022] Item 7: the method of item 6, wherein the mixture is a mixture of surfactant A and surfactant B having a negative Cc value, for example, surfactant B is a nonionic surfactant having a negative Cc value, for example surfactant B is a nonionic ethoxylated surfactant having a negative Cc value, for example surfactant B is a nonionic polysorbate having a negative Cc value, for example surfactant B is polyoxyethylene (20) sorbitan monooleate.

[0023] Item 8: the method of any one of items 0 to 7, wherein the mass of surfactant A represents less than 70%, for example between 50% and 70% of the mass of the surfactant mixture.

[0024] Item 9: the method of item 8, wherein the rest of the surfactant mixture is surfactant B.

[0025] Item 10: the method of any one of items 0 to 9, wherein the aqueous phase is devoid of alcohols of formula R-OH, wherein R is a hydrocarbon chain.

[0026] Item 1 1 : the method of any one of items 0 to 10, wherein the surfactant is dissolved in the oil phase before step c).

[0027] Item 12: the method of any one of items 0 to 1 1 , wherein the amount of surfactant is of 5 to 200 g / L of the oil phase, for example of 10 to 100 g / L of the oil phase.

[0028] Item 13: the method of any one of items 0 to 12, wherein the amount of surfactant is of 0.3 to 7.5 g / mL of the aqueous phase, for example of 0.5 to 5 g / mL of the aqueous phase.

[0029] Item 14: the method of any one of items 0 to 13, wherein the cross-linking agent is added to the aqueous phase before step c), for example between 1 and 6 minutes before step c).

[0030] Item 15: the method according to any one of items 0 to 14, wherein the cross-linking results in the formation of phosphodiester bonds between the polysaccharide chains.

[0031] Item 16: the method of item 15, wherein the cross-linking agent is trisodium trimetaphosphate (STMP).

[0032] Item 17: the method of any one of items 0 to 16, wherein the cross-linking agent is added to the aqueous phase before step c) in an amount of 5 to 100 g / 100 g of polysaccharide, for example of 10 to 50 g / 100 g of polysaccharide.

[0033] Item 18: the method of any one of items 0 to 17, wherein, a base is added to the aqueous phase before step c).

[0034] Item 19: the method of any one of items 0 to 18, wherein the base is added before the cross-linking agent.

[0035] Item 20: the method of item 18 or 19, wherein the base is a strong base. Item 21 : the method of item 20, wherein the strong base is NaOH.

[0036] Item 22: the method of any one of items 18 to 21 , wherein the pH of the aqueous phase is of at least 10, for example at least 1 1 , for example at least 12, for example at least 13.

[0037] Item 23: the method of any one of items 0 to 22, wherein, after adding the cross-linking agent, the temperature is kept below 20°C and wherein cross-linking comprises maintaining the emulsion at a temperature of 30°C to 90°C for at least 10 minutes.

[0038] Item 24: the method of item 23, wherein cross-linking comprises maintaining the emulsion at a temperature of 40°C to 80°C, for at least 15 minutes.

[0039] Item 25: the method of any one of items 0 to 24, wherein the concentration of the salt in the aqueous phase is of at least 1 mol / L, for example of 2 to 10 mol / L.

[0040] Item 26: the method of any one of items 0 to 25, wherein the salt is a monovalent salt.

[0041] Item 27: the method of any one of items 0 to 25, wherein the salinity of the aqueous phase is of 10 to 30 gNaci / 100 mL of the solution.

[0042] Item 28: the method of any one of items 0 to 27, wherein the volume of the aqueous phase at step c) represents 1 to 10% of the volume of the oil phase at step c).

[0043] Item 29: the method of any one of items 0 to 28, wherein the solvent of the oil phase is a vegetable oil.

[0044] Item 30: the method of item 29, wherein the vegetable oil is sunflower oil.

[0045] Item 31 : the method of any one of items 0 to 30, wherein the concentration of the polysaccharide in the aqueous phase at step a) is of 60 g / L to 1500 g / L, for example of 100 to 900 g / L.

[0046] Item 32: the method of any one of items 0 to 31 , wherein the polysaccharide consists of only one polysaccharide.

[0047] Item 33: the method of any one of items 0 to 31 , wherein the polysaccharide consists of a mixture of polysaccharides.

[0048] Item 34: the method of any one of items 0 to 33, wherein the polysaccharide comprises dextran.

[0049] Item 35: the method of item 34, 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. Item 36: the method of any one of items 0 to 35, wherein the polysaccharide comprises fucoidan.

[0050] Item 37: the method of any one of items 0 to 36, wherein recovering the particles comprises separating the particles from the oil phase by at least one centrifugation step, wherein after the centrifugation step, the supernatant is discarded, and the particles are resuspended in an aqueous solution.

[0051] Item 38: the method of any one of items 0 to 37, further comprising a step of loading the particles with a water-soluble active compound, for example a water-soluble drug, for example a fibrinolytic agent or a thrombolytic agent.

[0052] Item 39: the method of item 38, wherein the loading comprises: suspending the particles in an aqueous solution of the active compound to obtain particles loaded with the active compound, and recovering the loaded particles.

[0053] Item 40: the method of item 38 or 39, wherein the active compound is a protein.

[0054] Item 41 : the method of item 40, wherein the protein is a globular protein.

[0055] Item 42: the method of item 40 or 41 , wherein the protein has a mass of 10000 Da to 200000 Da.

[0056] Item 43: the method of any one of items 40 to 42, wherein the protein is a tissue-type plasminogen activator (tPA) or a deoxyribonuclease I (DNase I).

[0057] Item 44: the method of any one of items 40 to 43, wherein the loading comprises: suspending the particles in an aqueous solution of the protein to obtain particles loaded with the protein, and recovering the loaded particles, and wherein the pH of the aqueous solution of the protein is below the isoelectric point of the protein.

[0058] Item 45: particles obtainable by the method of any one of items 0 to 44.

[0059] Item 46: particles comprising a cross-linked polysaccharide matrix, having a hydrodynamic size of below 500 nm, for example of 150 nm to 400 nm, as measured by Dynamic Light Scattering in a 0.9 wt% NaCI in water solution.

[0060] Item 47: the particles of item 46, further characterized in that they are spherical. Item 48: the particles of item 46 or 47, further characterized in that they have a polydispersity index of below 0.2, as measured by Dynamic Light Scattering in a 0.9 wt% NaCI in water solution.

[0061] Item 49: the particles of any one of items 46 to 48, further characterized in that they have a Zeta potential of -40 to -20 mV for a pH of 4 to 10, for example for a pH of 2 to 12.

[0062] Item 50: the particles of any one of items 46 to 49, further comprising a releasable water-soluble active compound, for example a water-soluble drug, for example a fibrinolytic agent.

[0063] Item 51 : the particles of item 50, wherein the active compound is a protein.

[0064] Item 52: the particles of item 51 , wherein the protein is a globular protein.

[0065] Item 53: the particles of item 50 or 51 , wherein the protein has a mass of 10000 Da to 200000 Da.

[0066] Item 54: the particles of any one of items 51 to 53, wherein the protein is a tissue-type plasminogen activators (tPA), a deoxyribonuclease I (DNase I), or a combination thereof.

[0067] Item 55: the particles of any one of items 46 to 54, wherein the polysaccharide consists of only one polysaccharide.

[0068] Item 56: the particles of any one of items 46 to 54, wherein the polysaccharide consists of a mixture of polysaccharides.

[0069] Item 57: the particles of any one of items 46 to 56, wherein the polysaccharide comprises dextran.

[0070] Item 58: the particles of item 57, 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.

[0071] Item 59: the particles of any one of items 46 to 58, wherein the polysaccharide comprises fucoidan.

[0072] Item 60: the particles of any one of items 46 to 59, wherein the polysaccharide molecules are crosslinked via phosphodiester bonds.

[0073] Item 61 : the particles of item 60, comprising a phosphorus content of 5 to 15 pg / mg of particle.

[0074] Item 62: the particles of any one of items 50 to 61 , wherein the active agent is a water-soluble drug for use in a method of treatment of the human or animal body by therapy or a pharmaceutical composition comprising the particles of any one of items 50 to 61 , wherein the active agent is a water-soluble drug. Item 63: the particles for use of item 62 wherein the water-soluble drug is a fibrinolytic agent or a thrombolytic agent, preferably wherein the water-soluble drug is a tPA, a DNase I, or a combination thereof, and wherein the method of treatment is a method of treatment of a thrombosis by thrombolysis or the pharmaceutical composition of item 62, wherein the water-soluble drug is a tPA, a DNase I, or a combination thereof.

[0075] Item 64: 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 of item 63.

[0076] Brief description of the drawings

[0077] Fig. 1 Stability of nanoparticles of the present invention (Final protocol) compared to submicroparticles (Initial protocol) as disclosed in the prior art. Both samples were subjected to solvent exchanges and size and PDI were verified by DLS after 6 h. N = 3 batches per condition.

[0078] Fig. 2 Z-average hydrodynamic diameter and polydispersity (PDI) determined by DLS for nanoparticles of the present invention (Final protocol) compared to submicroparticles (Initial protocol) as disclosed in the prior art. Each triangle represents a different production batch.

[0079] Fig. 3 In flow distribution profile comparison of fucoidan containing 850 nm particles, obtained with the submicroparticles (Initial protocol) as disclosed in the prior art, with 315 nm Fuco-NPs (final protocol). The blood containing 1 mg / mL TRITC-tagged particles is flown at a venous flow rate in a microfluidic channel, and the fluorescence signal across a cross-section of the channel is measured. The X-axis corresponds to the cross-section distance (n=5).

[0080] Fig. 4 (Left) Loading of rtPA in particles of the invention determined by BCA (BiCinchoninic acid Assay). (Right) Release of rtPA after 1 h incubation at 37°C in PBS 1 X determined by BCA.

[0081] Fig. 5 (Left) Fibrin agar plate assay (FAPA) representative picture after 6 h. (Right) Amount of active rtPA loaded in nanoparticles of the present invention quantified by the FAPA assay.

[0082] Fig. 6 (Upper panel) Representative in flow targeting under arterial shear stress of platelet aggregates (green) by nanoparticles of the present invention (red). (Lower panel) Colocalization of empty or rtPA- loaded nanoparticles of the present invention and aggregates from inflow targeting assays measured by Pearson’s coefficient.

[0083] Fig. 7 Static thrombolysis with in vitro “young” clots. The released hemoglobin after 37°C incubation for 1 h was quantified with the Drabkin’s reagent and normalized to the initial clot mass. A one-way ANOVA with Tukey’s multiple comparison test was performed (n=4). Fig. 8 shows static thrombolysis with in vitro “old” clots. The released hemoglobin after 37°C incubation for 1 h was quantified with the Drabkin’s reagent and normalized to the initial clot mass. . A one-way ANOVA with Tukey’s multiple comparison test was performed (n=4).

[0084] Fig. 9 shows in vivo targeting of nanoparticles in a murine mesenteric vein thrombosis model. The fluorescence ratio of TRIC-labeled NPs with DIOC6 was compared for non-loaded Dex-NPs and Fuco- NPs, with saline injection as a control. A one-way ANOVA with Tukey’s multiple comparison test was performed (Saline, n=10; Dex-NPs, n=4; Fuco-NPs, n=10).

[0085] Fig. 10 compares thrombus area evolution for different treatments in a murine mesenteric vein thrombosis model. A one-way ANOVA with Tukey’s multiple comparison test was performed (from left to right, n=10; n=10; n=4; n=7; n=10; n=9; n=10; n=10; n=10). The thrombi which departed during the procedure were excluded from the analysis.

[0086] Fig. 1 1 (Left) compares DNase I loading capacity in Dex-NPs and Fuco-NPs by BCA ([DNase l]=0.5 mg / mL, [NP]=2.5 mg / mL). An unpaired t-test was performed. (n=3). Fig. 1 1 (Right) displays NETolysis assay. Neutrophils isolated from human blood were seeded then activated with PMA to release NETs, followed by treatment with DNase I or Fuco-NP-DNase. DAPI and SytoxGreen were used to differentially stain intra- and extracellular DNA. Top left: neutrophils not exposed to PMA and treatments. Top right: NETs released with PMA but treated with PBS. Bottom left and right: NETs released by PMA and treated with respectively 0.5 mg / mL DNase I or equivalent DNase I concentration in Fuco-NP-DNase (experiment repeated three times) (scale bar = 200 pm).

[0087] Detailed description

[0088] The method of the invention relies on the selection of a particular mix of surfactants that allows the formation of a suitable emulsion, that in its turn yields particles comprising a cross-linked polysaccharide matrix with a smaller size than in the prior art. Moreover, it has been observed that the size of the particles does not vary depending on the ionic strength of the aqueous solution in which they are dispersed and that the particles are stable in a saline aqueous solution even in the absence of added surfactant. Moreover, the particles are reliably obtained with a low polydispersity.

[0089] Hydrophilic-lipophilic difference (HLD)

[0090] As defined herein, the term emulsion encompasses both unstable and metastable emulsions and microemulsions. Microemulsions are thermodynamically stable emulsions.

[0091] HLD, the hydrophilic-lipophilic difference, is a well-known parameter in the art of emulsions. Systems with positive HLD generally form w / o emulsions, whereas systems with negative HLD generally form o / w emulsions. Moreover, systems having HLD close to 0 can form microemulsions. More detailed information about the HLD can be found, for example, in the book Surfactant Science: Principles and Practice, by Prof Steven Abbott, available online at https: / / www.stevenabbott.co.uk / practical-surfactants / the-book.php or in Salager, Jean-Louis, Anton, Raquel, Anderez, Jose, Aubry, Jean-Marie. (2001 ). Formulation des micro-emulsions par la methode HLD. Techniques de I'lngenieur. 1 -20. DOI:10.51257 / a-v1 -j2157.

[0092] The HLD can be calculated as follows:

[0093] HLD = F(S) - k.EACN - a(T-25) + Cc,

[0094] Wherein:

[0095] S is the salinity of the aqueous phase expressed in gNaci 1 100 mL of aqueous phase. Expressing the salinity in gNaci / 100 mL means for example that, for monovalent salts S = rrisait x (58 / Msait) g 1 100 mL. For divalent and trivalent salts the value of S is changed via the ionic strength according to corrections known of the skilled person (equivalent S for multivalent salts can be calculated e.g. at this URL: https: / / www.stevenabbott.co.uk / practical-surfactants / hld-expert.php).

[0096] In the present disclosure, S is calculated for a hypothetical solution that comprises the same salt and amount of salt as the aqueous phase but ignoring the contribution of the base, the cross-linking agent and of the polysaccharide.

[0097] F(S) = 0.13 x S when the surfactant is nonionic and F(S) = ln(S) when the surfactant is ionic.

[0098] EACN, the Effective Alkane Carbon Number, is characteristic of the oil. EACN values are tabulated or can be measured, for example according to the procedure below.

[0099] The EACN scaling factor, k, is characteristic of the surfactant. It is tabulated or can be measured.

[0100] T is the temperature. a is characteristic of the surfactant, a values are tabulated.

[0101] In the present disclosure, the HLD is calculated with T taken to be 25°C, so that the temperature term vanishes.

[0102] Cc, the Characteristic Curvature, is characteristic of the surfactant. Cc values are tabulated or can be measured, for example according to the procedure below.

[0103] EACN determination EACN of an oil is determined by screenings of tubes with varying Cc. Centrifugation tubes are set as Cc screening range with varying mix of surfactants with known Cc. 750 gL of the oil is mixed with 90 mg surfactant mix for one hour. Then, 750 gL ultrapure water is gently added, and followed by three quick inversions by hand. Then the tubes are set to rest at RT for at least 2 h. Visual inspection allows to identify which tubes contained a o / w emulsion (phase I), a w / o emulsion (phase II), or a microemulsion (phase III). The EACN value for the oil is determined as the value that would result in the same pattern of tubes. This can be repeated with various known surfactants depending on the accuracy needed.

[0104] Cc determination

[0105] Cc of a surfactant is determined by screenings of tubes with varying Cc. Centrifugation tubes are set as Cc screening range with varying mix of a surfactant with known Cc and the surfactant whose Cc is determined. 750 gL of an oil of known EACN is mixed with 90 mg surfactant mix for one hour. Then, 750 gL ultrapure water is gently added, and followed by three quick inversions by hand. Then the tubes are set to rest at RT for more than 2 h. Visual inspection allowed to identify which tubes contained a o / w emulsion (phase I), a w / o emulsion (phase II), or a microemulsion (phase III). The Cc value for the surfactant is determined as the value that would result in the same pattern of tubes. This can be repeated with various known surfactants depending on the accuracy needed.

[0106] Surfactant

[0107] As defined herein, and unless specified otherwise, a surfactant can be a mixture of surfactants.

[0108] Surfactants with negative Cc values are more hydrophilic surfactants, whereas surfactants with positive Cc values are more lipophilic.

[0109] The Cc of a mixture of surfactants can be computed from the parameter Cc / k of the mixture. The Cc of a mixture of surfactants over the k of said mixture is the sum of the Cc / k of the surfactants in the mixture weighted by their molar fraction in the mixture of surfactants and the k a mixture of surfactants is the sum of the k of the surfactants in the mixture weighted by their molar fraction in the mixture of surfactants.

[0110] An example of a surfactant A comprising a polyethyleneglycol chain and a polyester of a monohydroxylated fatty acid is PEG-30 dipolyhydroxystearate.

[0111] As defined herein, an ethoxylated surfactant is a surfactant whose hydrophilic part comprises polymers or oligomers of ethylene glycol (the repeated unit is -CH2-CH2-O-).

[0112] Examples of nonionic polysorbate having a negative Cc value comprise Tween 60 (polyoxyethylene (20) sorbitan monostearate) and Tween 80 (polyoxyethylene (20) sorbitan monooleate). Alcohols are often used as co-surfactant for formulating emulsions. However, the presence of alcohols can lead to the precipitation of the polysaccharide.

[0113] Oil phase

[0114] The oil phase is a hydrophobic and lipophilic liquid phase

[0115] Cross-linking agent

[0116] The timing of the addition of the cross-linking agent and the control of the temperature of the mix is useful to avoid unwanted cross-linking before the cross-linking step d).

[0117] Examples of crosslinking agents that result in the formation of phosphodiester bond between the polysaccharide chains comprise STMP or POCI3.

[0118] A phosphodiester bond liking two groups R has the following formula: R-O-P(=O)(-Oj-O-R.

[0119] The base facilitates the cross-linking of the polysaccharide.

[0120] Salt

[0121] 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 chains in the presence of 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.

[0122] 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 .

[0123] Polysaccharide

[0124] As defined herein, and unless specified otherwise, a polysaccharide can be a mixture of polysaccharides.

[0125] 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.

[0126] Particles comprising a cross-linked polysaccharide matrix, such as the particles of the present disclosure, are often referred to as hydrogel particles.

[0127] Active agent As defined herein, an active compound can be a drug or a contrast agent.

[0128] As defined herein, tissue type plasminogen activator and deoxyribonuclease I comprise recombinant tissue type plasminogen activator and recombinant deoxyribonuclease I.

[0129] As defined herein, particles of the invention comprising a combination of a DNase I and a tPA comprise mixtures of particles comprising a DNase I with particles comprising a tPA.

[0130] For negatively charged particles, lowering the pH of the aqueous solution of the protein below the isoelectric point of the protein favors the loading of the protein.

[0131] 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.

[0132] Characteristics of the particles

[0133] The hydrodynamic size (also called Z-average size) is measured in a 0.9 wt% NaCI in water solution by dynamic light scattering (DLS), for example using an instrument such as a Zetasizer Nano ZS (Malvern Instruments, 30 Orsay, France).

[0134] A 0.9 wt% NaCI in water solution designates a solution comprising only water and the 0.9wt% NaCI. A 0.9 wt% NaCI in water solution does not comprise a surfactant.

[0135] The hydrodynamic size and the polydispersity index (PDI) are defined by the ISO norms: ISO 22412:2017 and ISO 22412:2017.

[0136] Particles obtained by an emulsion method are generally spherical since surface tension imposes a spherical shape for the droplets of the emulsion used to produce the particles.

[0137] Zeta potential is measured by Electrophoretic Light Scattering (ELS), for example using a Zetasizer Nano ZS (Malvern Instruments, Orsay, France). Samples are diluted in 1 mM KCI.

[0138] Zeta potential is defined by ISO norms ISO 13099-2:2012 and ISO 13099-1 :2012. A releasable compound is a compound that is not covalently linked to the particle and that can be released from the particle when the particle is diluted in physiologically relevant conditions, for example in phosphate-buffered saline (PBS 1 X) at 37°C.

[0139] Examples

[0140] Materials

[0141] Dextran 40 kDa was purchased from Pharmacosmos (Holbaek, Danemark, #5510 0040 1007); FITC- Dextran 40 kDa and TRITC-Dextran 40 kDa were purchased from TDB Consultancy (Uppsala, Sweden, #FD40 and #TD40). Fucoidan (Mn = 18 kDa / Mw = 104 kDa) was a gift from Algues & Mer (Ouessant, France, #MMWFSA14093). Vegetable sunflower oil (Lesieur - Huile Coeur de Tournesol, Lesieur S.A.S, Asnieres-sur-Seine, France) was purchased from a local supermarket (Monoprix, Paris, France). Polyglycerol polyricinoleate (PGPR) was kindly supplied by Palsgaard (Lyon, France, # PGPR 4150). Fetal bovine serum (FBS) was purchased from Pan-Biotech (Aidenbach, Germany, #P30-3306). HUVEC cells line (ATCC, Manassas, Virginie, #ATCC-CRL-1730) was used at fewer than 40 passages. Tween 80 (#P1754), Trisodium trimetaphosphate (STMP, #T5508), Sodium Dodecyl Sulfate (SDS, #L3771 ), Sucrose (#S7903), fibrinogen type I from human plasma (#F3879), thrombin from human plasma (#T7009), plasminogen from human plasma (#528175) and Iron (III) Chloride hexadydrate (FeC , #236489-100G) were purchased from Sigma Aldrich (Saint-Quentin-Fallavier, France). DMEM, low glucose, pyruvate (#31885049), Antibiotic Antimycotic solution (#15240062), PBS 1 X and 10X (#14040133 and #14080055), Rhodamine phalloidin (#R415), low melting point agarose (#R0801 ), DIOC6 (D273), DAPI (#62247), SytoxGreen (#S7020) were purchased from Thermo Fisher Scientific (Massachusetts, United States). Horm® collagen type I was purchased from Takeda Pharmaceutical (Tokyo, Japan). rtPA (alteplase) was purchased from Boehringer Ingelheim (Ingelheim am Rhein, Germany, # Actilyse®). DNase I (dornase alfa) was purchased from Roche - Genentech (San Francisco, United States, #Pulmozyme® 2500 U / 2,5 mL). The DNase I detection kit (#JE PP 410L) was purchased from Euromedex (Souffelweyersheim, France). Polymorphprep (#1 1 14683) was purchased from Proteogenix (Schiltigheim, France). Human whole blood was acquired from EFS (Bichat Hospital, Paris, France), the French blood donation institute, under a research convention, in PPACK, sodium citrate or EDTA collection tubes.

[0142] Methods

[0143] Nanoparticle synthesis according to the invention: polysaccharide nanoparticles (NPs) were obtained through a water-in-oil (w / o) microemulsion, with chemical crosslinking in the aqueous phase. A polysaccharide solution (300 g / L polysaccharide, 6 M NaCI) was prepared; The polysaccharide was either pure Dextran 40 or Dextran 40 in a 90% w / w ratio with fucoidan at a 10% w / w ratio. For fluorescent NPs, a 5:95 mass mixture of TRITC-Dextran 40 or FITC-Dextran 40 with Dextran 40 was used. 15 mL organic phase composed of sunflower oil and 6% w / v surfactant (60:40 PGPR and Tween 80) was cooled at -20°C for 30 min. Meanwhile, 1200 mg of the polysaccharide solution is mixed with 60 pL of 10 M NaOH for 10 min. 240 pL of 30 % w / v STMP solution was added before quick mixing for 1 min. 600 |aL of this aqueous mix are then added dropwise to the organic phase under dispersion with a standdisperser (Polytron PT 3100, dispersing aggregate PT-DA 07 / 2 EC-B101 , Kinematica, Luzern, Switzerland) for 2 min 30 s at 30’000 rpm on ice. The resulting emulsion was incubated at 50°C for 20 min before successive purification by ultracentrifugation (50’000 g, 30 min) then resuspending the pellets with SDS 0.04 % w / v four times, then with ultrapure water three times. The resulting NPs were stored at 4°C in ultrapure water. Concentration of NPs could be performed with ultracentrifugation devices with size cutoff of 300’000 Da (Vivaspin, Dutscher, Issy-les-Moulineaux, France). Stable freeze-dried NPs were obtained with the LyoVac GT2 freeze dryer (SRK Systemtechnik, Riedstadt, Allemagne) after -20 °C freezing for 1 h in a 1 % sucrose solution. Notably, stable freeze-dried NPs were obtained for rtPA loaded NPs. Pure Dextran NPs and Fucoidan-containing NPs are respectively abbreviated Dex-NPs and Fuco-NPs.

[0144] Comparative submicroparticle synthesis: Polysaccharide submicroparticles (SPs) were obtained via a water-in-oil (w / o) emulsification combined with a crosslinking process. Polysaccharide solution (300 mg / ml, 6 M NaCI) was prepared. The polysaccharide was Dextran 40 in a 90% w / w ratio with fucoidan at a 10% w / w ratio. The same procedure as for the NPs is used except that the surfactant was PGPR only (dissolved in the organic phase at a concentration 6% w / v).

[0145] HLD calculations

[0146] A Cc of 4 was determined for PGPR. For Tween 80, Cc is tabulated at -3.7.

[0147] A k of 0.16 is taken both for PGPR and for Tween 80.

[0148] EACN for sunflower oil was determined at 16.

[0149] Both for the NPs and for the SPs, the salinity (ignoring the STMP and any charged polysaccharide) is of 18.8 gNaci / 100 mL of aqueous phase.

[0150] For the NPs, the molar fraction of PGPR is 0.79 (taking a molar mass of 521 g / mol for PGPR) and the molar fraction of Tween 80 is 0.21 (taking a molar mass of 1310 for Tween 80).

[0151] HLDNP = 0.13*18.8 - 0.16*16 + 0.79*4 + 0.21 *(-3.7) = 2.27

[0152] HLDSP = 0.13*18.8 - 0.16*16 + 4 = 3.88

[0153] Physico-chemical characterization: The particles formulations were studied for particle morphology, size and zeta potential distributions, mass concentration, and elemental composition. Particle morphology was visualized by Transmission Electron Microscopy (TEM) (Philips FEI Tecnai 12, Amsterdam, Netherlands), negatively stained with 1 % (w / v) uranyl acetate for 5 min. Hydrodynamic size (diameter), polydispersity (PDI) 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 diluted in distilled water, 0.9% NaCI or PBS 1 X for size, and in 1 mM KCI for ^-potential determination. All runs were performed at 25°C in triplicate. Mass concentration was determined by freeze-drying. Elemental composition was assessed by total reflection X-ray fluorescence spectroscopy (TXRF) technique to quantify the phosphorus and sulfur content (S2 PICOFOX Bruker, Massachusetts, United States). The stability of Fuco-NPS and compared submicroparticles (SPs) in various solvents (ultrapure water, 0.9% NaCI, 0.9% NaCI and 0.02% Tween 20) was evaluated 6 h after solvent exchange.

[0154] Sulfate and fucoidan quantification: The sulfate content of fucoidan was determined by a semi- quantitative solid-phase colorimetric assay. Briefly, 12.5 pg of Fuco-NPs in suspension at a concentration of around 1 mg / mL were added by 5 pL drops on a piece of Whatman Chromatography paper grade 1 with drying between each drop addition. The paper was first soaked into a methanol / acetone (6:4) solution for 3 min and then into a methanol / acetone / water (6:4:15) solution with 50 mM HCI and 0.1 % w / w methylene blue for 10 min. Finally, the paper was extensively washed with acetic acid / methanol / acetone / water (5:6:4:85) until no coloration was detected in the washing solution. The paper was then transferred to the Eppendorf, containing 0.5 mL methanol with 2% w / v SDS, and incubated for 15 min at 50°C. 0.2 mL of the extracted dye was placed in a 96-well plate, and its concentration was determined by reading absorbance at 663 nm with a Varioskan LUX multiplate reader (Thermo Fisher Scientific, Massachusetts, United States). Standard curves were obtained from fucoidan in solution with known concentrations.

[0155] Cell culture and cytotoxicity assay: To evaluate the cytotoxicity of the SPs, Fluorometric Cell Viability Assay (#TOX-8 kit, Sigma Aldrich, Saint-Quentin-Fallavier, France) was used on confluent Human Umbilical Vein Endothelial Cells (HUVECs). The cells were cultured in low glucose DMEM supplemented with 10 % (v / v) FBS and 1 % antibiotic-antimycotic; the cells were kept in an incubator at 37°C in a humidified atmosphere of 5 % CO2. Cells were seeded into 96-well plates with 30,000 cells per well. Following 24 h of incubation, the medium was changed to Fuco-NPs resuspended in culture medium in a range of 100 mg / L to 1 g / L, and the cells were cultured for another 24 h. Cells cultured with equivalent PBS 1 X medium dilution were set as control. Negative controls were culture medium only, and positive controls were 1 % Triton X-100 in culture medium for 15 min. Then the medium was replaced with 100 pL 10 % Resazurin solution in culture medium, and the plates were covered in foil and incubated for another 2 h. The Resazurin ’s absorbance signals were monitored at 570 nm and 590 nm wavelengths with a Varioskan LUX multiplate reader. The corrected absorbance values were blank corrected: A’ = As / o nm - Asgo nm, and the relative cell viability was expressed as (A’ - A’neg) / (A’pos - A’neg) x 100 %, with the negative and positive control absorbances respectively averaged in the control wells as detailed above. To examine possible cell morphology changes after co-incubation with Fuco-NPs, HUVECs cells were seeded in 8-wells Lab-Tek II Chamber Slide w / Cover (Thermo Fischer Scientific, Massachusetts, United States) with 10,000 cells per well. The medium was changed 24 h to Fuco-NPs resuspended in culture medium in a range of 100 mg / L to 1 g / L, and the cells were cultured for another 24 h. Cells cultured with equivalent PBS 1 X medium dilution were set as control. Next, cells were fixed with 4% paraformaldehyde for 30 min at 4°C. After rinsing with PBS, cells were permeabilized with T riton X-100 0.1 % in PBS for 5 min at room temperature (RT), then washed twice with PBS before incubation for 60 min at RT with 1 % Phalloidin-Rhodamine in PBS. After two PBS washes, the slides are mounted with a Mounting Medium with DAPI. Visualization was performed with a confocal microscope (Zeiss LSM 780, lena, Germany).

[0156] Drug loading: rtPA was loaded onto the NPs by adsorption. 50 pL of NPs (5 mg / mL) were mixed with 50 pL rtPA (1 mg / mL) in ultrapure water then incubated for 15 min. Free unabsorbed rtPA was removed by ultracentrifugation (15 min, 15’000 g). The rtPA loaded NPs were resuspended in ultrapure water, and rtPA encapsulation was determined as described below by BOA and PefaFluor® tPA assays. DNase I was loaded onto the NPs by adsorption in acidic conditions. 50 pL of NPs (5 mg / mL) were mixed with 25 pL DNase I (1 mg / mL), 5 pL of HCI (2x102M) and 20 pL ultrapure water (or 10 pL CaCh (650 pM) and 10 pL ultrapure water) then incubated for 15 min. Free unabsorbed DNase I was removed by ultracentrifugation (15 min, 15’000 g). The DNase I loaded NPs were resuspended in ultrapure water and DNase I encapsulation was determined as described below by BOA and a fluorescent substrate DNAse detection kit assays.

[0157] Drug encapsulation efficiency: The amount of drug (rtPA or DNase I) loaded on the NPs was measured using the Pierce BCA protein assay kit (Life Technologies SAS, Courtaboeuf, France). Briefly, 200 pL of working reagent was added to 25 pL of each sample in 96 wells multiplate. The absorbance at 562 nm was read with a Varioskan LUX multiplate reader after 30 min incubation at 37°C. The concentration of the drug was extrapolated by a calibration curve prepared with different concentrations of rtPA or DNase I respectively. The encapsulation efficacy (EE) was calculated as EE (%) = 100% x [Drug]ioaded / [Drug]initiai and the loading capacity (LC) was calculated as LC (%) = 100% x [Drug]ioaded / [NP],

[0158] Fibrin Agar Plate Assay (FAPA): To assess the fibrinolytic activity of rtPA-loaded NPs, a fibrinolysis experiment was performed. 5 mL of TBS Buffer (0.1 M Tris, 0.8 % NaCI, 0.02 % KOI, pH = 7.4) with 3% w / v low melting agarose were heated above 65°C. 5 mL of TBS buffer with 25 mg fibrinogen and 1 U plasminogen was slowly heated to 37°C. Once the agarose solution reached 65 °C, it was cooled to 37°C, and 2 U of thrombin were added. Next, the two solutions were slowly mixed, then poured into a 9 cm Petri dish and incubated at 37°C for 2 h (gels can be kept a few days at 4°C). On the solidified agarose gel, round wells were formed using a 3 mm punch as sample reservoirs. 5 pL of each NPs sample (diluted 1 :10) was dropped into the wells and incubated at 37°C in a humid environment. Pictures were taken every hour for 8 h. The degree of fibrin lysis was quantified with Imaged by comparing the rate of fibrinolysis between samples and free rtPA standards.

[0159] In flow platelet aggregates targeting assay: An in vitro flow adhesion assay was performed to evaluate the targeting of Fuco-NPs with to activated platelets. Micro-channels of Vena8 Fluoro+chambers (width: 0.04 cm, height: 0.01 cm, and length: 2.8 cm; Cellix Ltd, Dublin, Ireland) were coated overnight with fibrillar type I collagen (50 pg / ml) 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 5 pM DIOC6 (to image platelets), 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 Dex-NPs or Fuco-NPs (unloaded or loaded with rtPA) resuspended in 0.9 % NaCI at 1 mg / mL, were injected into the channels for 5 min. Their accumulation onto activated aggregates was monitored in real time. Channels were then washed for 5 min with NaCI 0.9%. Finally, quantification of bound NPs to aggregated platelets were performed with Imaged by measuring total fluorescence intensity and colocalization with the Pearson’s coefficient.

[0160] Flow distribution assay: an in vitro blood flow assay was performed to evaluate the margination of NPs within blood flow. Micro channels of Vena8 Fluoro+ chambers (width: 0.04 cm, height: 0.01 cm, and length: 2.8 cm; Cellix Ltd, Dublin, Ireland) were rinsed with NaCI 0.9 wt.% before use. Human whole blood was collected in sodium citrate tubes and labeled with 5 pM DIOC6, then NPs were added to a concentration of 1 mg / mL. The blood was then perfused at venous shear stress (here, 7 pL / min) for 2 min and fluorescence images were acquired with a microscope (Axio Observer, Carl Zeiss Microscopy, Oberkochen, Germany). Fluorescence was measured across 5 cross-sections per channel and averaged over 2 min for each sample.

[0161] In vitro static thrombolysis: for static thrombolysis assays, two types of blood clots were prepared in vitro: “young” and “old” clots. Human whole blood was collected in sodium citrate tubes, and 200 pL blood were added to glass tubes containing 10 pL water with 200 mM CaCh and 2 U / mL thrombin. The tubes were closed and incubated at 37°C for 1 h for “young” clots. For “old” clots, 0.2 U / mL thrombin and 24 h incubation were used. The resulting clots were washed four times in PBS, weighed, then washed again three times. The clots were then resuspended in PBS, with 0.25 U plasminogen added per clot and incubated at 37°C for 1 h with slow agitation. The clots were weighed again, and the supernatants were frozen at -20°C for 24 h. Quantification of released hemoglobin (Hb) was performed by thawing the supernatants then mixing twenty 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 = 7.4) with one volume supernatant. After 10 min incubation in the dark at RT, absorbance is read at 540 nm with a Varioskan LUX multiplate reader (Thermo Fisher Scientific, Massachusetts, United States). rtPA and DNase I concentrations used were 1 pg / mL and 50 pg / mL respectively , whether the active agent was added in free form or loaded in the particles of the invention . DNase was only tested with “old” clots.

[0162] In vivo FeCIg mesenteric vein thrombosis model: animal studies were performed on 6-8 weeks old C57BL6J mice (Janvier Labs, Le Genest Saint Isle, France), with equipartition of males (24.9±2.3 g weight) and females (19.9±2.4 g weight) in each group. All experiment were adapted according to French (Decree 87 / 848) and European (2010 / 63 / EU) ethical guidelines. The local ethic committee (C2EA 121 Paris Nord, France) and the French Ministere de I’Enseignement Superieur et de la Recherche approved the fulfilling of the experiments with the reference number APAFIS#49260. 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.% FeCh 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, images were acquired at 20 s intervals for 30 min. Between the second and third frames, samples were administered with a retro orbital injection (150 pL injections, in saline suspension). When NPs were used, they were fluorescently labeled with TRITC, and the maximum final concentration of Fuco-NPs in the mice blood was 962±26 pg / mL (obtained for the Fuco-NP-rtPA concentration such that the 5 mg / kg of rtPA were administered and for empty Fuco-NPs; as for the Fuco-NP-rtPA at 1 mg / kg, the total concentration of loaded Fuco- NPs was five times lower). At the end of the image acquisition, the animals were sacrificed. Image analysis of thrombi with ImageJ included a SIFT based alignment with rigid transformation. The thrombus area was measured over time in the DIOC6 channel to create thrombi binary masks by thresholding and the rate of thrombus degradation was analyzed after the thrombus reached its maximum size in the first 20 frames (7 minutes). For targeting measurements, the mean fluorescence intensity (MFI) was measured in the thrombus for both DIOC6 and TRITC over time, and in particular after 30 min as a ratio of MFITRITC / MFIDIOCB. The concentrations of rtPA expressed in mg / kg relate to mg of rtPA per kg of sample (i.e. the mice).

[0163] NET (Neutrophil Extracellular Traps) degradation assay: to assess the NETolytic activity of DNase I loaded NPs, a semi quantitative assay was performed. Human whole blood was collected in EDTA tubes. Following the supplier’s protocol for Polymorphprep, neutrophils were isolated and resuspended in 0.05 wt.% BSA in DMEM. Purity and cell count were determined with a hematology analyzer (ABX Pentra 60, Horiba, Venissieux, France). Neutrophils were seeded at 1 .0 x 105cells / cm2on 8 wells Lab Tek II Chamber Slide (Thermo Fischer Scientific, Massachusetts, United States) and incubated at a humidified atmosphere of 5% CO2 for 60 min. 4-phorbol-12-beta-myristate-13-acetate (PMA) was then added to a final concentration of 50 nM, and the neutrophils were left to incubate for 4 h. Medium was then changed to incubate the formed NETs with DNase I loaded Fuco NPs resuspended in 0.05 wt.% BSA DMEM to a concentration equivalent to 0.5 mg / mL or 0.1 mg / mL DNase I. After a 60 min incubation, the samples were fixed with 4% (v / v) paraformaldehyde for 30 min at RT. After rinsing three times with PBS, the samples were blocked with 1 % BSA in 0.1 % (v / v) Tween 20 PBS (PBS-T) for 30 min at RT, then washed with PBS three times. Then, the samples were incubated with 100 nM SytoxGreen and 1 pg / mL DAPI in 1 wt.% BSA PBS T for 10 min at RT, then washed with PBS twice before slide mounting and imaging with a microscope (Axio Observer, Carl Zeiss Microscopy, Oberkochen, Germany). Image analysis and statistical analysis: images were analyzed with Imaged (Fiji). When images are shown, the same contrast values are applied to each channel displayed. Quantitative data were analyzed with the statistical analysis software GraphPad PRISM 9.5.O., with a significance level a taken at 0.05. All presented data consisted of n=3 or more replicates per condition (when not shown on the graphs, replicate number is indicated in the legend). Whenever applicable, presented replicates consist of biological replicates, or, when appropriate, different NP batches. Unless explicitly mentioned, results are presented as mean ± standard deviation. Depending on the design of the experiments, data significance was evaluated with unpaired t test (two groups, assumed equal variance), unpaired t-test with Welch’s correction (two groups, variance not equal), paired t-test (two groups of paired values, with assumed equal variance), one way ANOVA (one factor explored, with more than two groups, variances assumed equal) with Tukey’s multiple comparison test, one way Welch’s ANOVA (one factor explored, with more than two groups, variances not equal) with Dunnett’s T3 multiple comparison test, repeated measures one way ANOVA (one factor explored, with more than two groups of repeated measures compared to the initial group, variances assumed equal) with Dunnett’s multiple comparison test, two- way ANOVA (two factors explored) with Tukey’s (comparison of all conditions) or Sidak’s (comparison between factors) multiple comparison test. Unless explicitly specified in the legend, unpaired t-tests and ANOVA with Tukey’s test are performed. Every p value below a threshold was represented as follows: * p < 0.05; “ p < 0.01 ; *** p < 0.001 , **** p < 0.0001 (ns was indicated for “not significant” if no significant differences are present in the data presented).

[0164] Results

[0165] Fig. 1 compares the size and stability in various solutions of the comparative SPs and of the Fuco-NPs of the invention. On the figure, “Initial protocol” designates the comparative SPs and “Final protocol” designates the Fuco-NPs of the invention. It can be seen that the comparative SPs have an increased PDI in 0.9% NaCI, which indicates that the particles were destabilized and that aggregation took place.

[0166] Fig. 2 compares the reliability of the synthesis of the comparative SPs and of the Fuco-NPs of the invention. Size and polydispersity were measured in pure water. It can be seen that the size and polydispersity of the comparative SPs is much more variable from batch to batch than the size and polydispersity of the Fuco-NPs of the invention.

[0167] Fig. 3 shows that the 850 nm particles display a clear margination pattern (the particles have an increased distribution near the borders of the channel) in the blood due to their size, while the smaller 315 nm nanoparticles show a homogeneous distribution across the channel.

[0168] Fig. 4 shows that the particles of the invention are able to load and release tPA efficiently.

[0169] Fig. 5 shows the efficiency of the particles of the invention as particles for drug delivery since there is no loss of fibrinolytic activity of the tPA that has been loaded and released. Fig. 6 shows that the addition of fucoidan in the particles of the invention helps targeting platelet aggregates and thus should allow thrombus targeting. Moreover, targeting capacity is retained when the Fuco-NPs are loaded with rtPA.

[0170] Fig. 7 shows that the thrombolytic potential of rtPA after loading in Fuco-NPs was similar to free rtPA in this static assay, demonstrating that the loading did not impair the fibrinolytic activity of rtPA.

[0171] Fig. 8 shows that while DNase I has no thrombolytic activity on its own, combination with rtPA improved the thrombolytic effect of the latter in a synergic manner. Moreover, loading of rtPA and DNase I in Fuco- NPs did not impair their thrombolytic activity.

[0172] Fig. 9 shows that the increased targeting of the thrombus for nanoparticles with fucoidan compared to the ones without it is clearly displayed, validating the in vivo thrombus targeting properties of Fuco-NPs.

[0173] Fig. 10 demonstrates that Fuco-NP loaded with rtPA at concentrations such that 5 mg / kg or 1 mg / kg of rtPA (i.e. 50% and 10% the recommended free rtPA dose in murine models of 10 mg / kg of rtPA) produced a similar thrombolysis as the free drug.

[0174] Fig. 1 1 (Left) demonstrates that DNase I can be loaded in the nanoparticles described in this invention. Fig. 1 1 (Right) validates that this loading conserved the loaded DNase I activity: the capacity of DNase

[0175] I to degrade NETs was retained.

Claims

CLAIMS1 . Method for the preparation of particles comprising a cross-linked polysaccharide matrix, said method comprising the following steps: a) providing an aqueous phase comprising the polysaccharide and a salt; b) providing an oil phase comprising an oil as a solvent; c) mixing the aqueous phase and the oil phase in presence of a surfactant so as to obtain a water / oil (w / o) emulsion; d) crosslinking the polysaccharide comprised in the dispersed aqueous phase to obtain the particles, wherein the crosslinking comprises the reaction between a crosslinking agent and the polysaccharide to obtain the polysaccharide matrix; e) recovering the particles; wherein the concentration of the salt in the aqueous phase, the oil, and the surfactant are selected so that the HLD at a temperature of 25°C is of 0 to 3, for example of 0 to 2.5, for example of 1 to 2.5.

2. The method of claim 1 , wherein the surfactant is a mixture of at least two different surfactants and the mixture of surfactants comprises a surfactant A, which is 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, one said surfactant A is polyglycerol polyricinoleate.

3. The method of claim 2, wherein the mixture is a mixture of surfactant A and a surfactant B having a negative Cc value, for example, surfactant B is a nonionic surfactant having a negative Cc value, for example surfactant B is a nonionic polysorbate having a negative Cc value, for example surfactant B is polyoxyethylene (20) sorbitan monooleate.

4. The method of claim 2 or 3, wherein the mass of surfactant A represents less than 60%, for example between 50% and 60% of the mass of the surfactant mixture.

5. The method of claim 4, wherein the rest of the surfactant mixture is surfactant B.

6. The method of claim 5, wherein the cross-linking results in the formation of phosphodiester bonds between the polysaccharide chains, for example the cross-linking agent is trisodium trimetaphosphate (STMP).

7. The method of any one of claims 1 to 6, wherein the volume of the aqueous phase at step c) represents 1 to 10% of the volume of the oil phase at step c).

8. The method of any one of claims 1 to 7, wherein the solvent of the oil phase is a vegetable oil, for example sunflower oil.

9. The method of any one of claims 1 to 8, wherein the polysaccharide comprises dextran, for example a dextran having a weight average molar mass of 10000 to 80000 g / mol, for example of 10000 to 60000 g / mol, for example of 35000 to 45000 g / mol.

10. The method of any one of claims 1 to 9, wherein the polysaccharide comprises fucoidan.1 1 . The method of any one of claims 1 to 10, further comprising a step of loading the particles with a water-soluble active compound, for example a water-soluble drug, for example a fibrinolytic agent or a thrombolytic agent.

12. Particles obtainable by the method of any one of claims 1 to 1 1 .

13. Particles comprising a cross-linked polysaccharide matrix, having a hydrodynamic size of below 500 nm, for example of 150 nm to 400 nm, as measured by Dynamic Light Scattering in a 0.9 wt% NaCI in water solution.

14. The particles of claim 13, further characterized in that they have a polydispersity index of below 0.2, as measured by Dynamic Light Scattering in a 0.9 wt% NaCI in water solution.

15. The particles of claim 13 or 14, further comprising a releasable water-soluble active compound, for example a water-soluble drug, for example a fibrinolytic or a thrombolytic agent.

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