Composition comprising a biocompatible polymer and at least one extracellular vesicle, and preparation method

Abstract

The present invention relates to a pharmaceutical composition comprising a biocompatible polymer of general formula (I) AaXxYy or (II) AaXxYyZz, and at least one extracellular vesicle. The invention also relates to a method for preparing extracellular vesicles. The present invention is applicable in particular in the therapeutic, pharmaceutical and veterinary fields.

Description

[0001] COMPOSITION COMPRISING A BIOCOMPATIBLE POLYMER AND AT LEAST ONE EXTRACT VESICULE AND PREPARATION METHOD

[0002] technical field

[0003] The present invention relates to a pharmaceutical composition comprising a biocompatible polymer of general formula (I) AaXxYy and at least one extracellular vesicle.

[0004] The present invention also relates to a pharmaceutical composition comprising a biocompatible polymer of general formula (II) AaXxYyZz and at least one extracellular vesicle.

[0005] The present invention also relates to a method for preparing extracellular vesicles and to the vesicles obtained by said method.

[0006] The present invention finds application particularly in the prophylactic, therapeutic, pharmaceutical fields for humans and veterinarians.

[0007] In the description below, references in brackets [ ] refer to the list of references presented at the end of the text.

[0008] State of the art

[0009] The implantation of cells, tissues or organs for therapeutic purposes, particularly the treatment of tissue damage, is a major challenge in medicine.

[0010] However, after cell administration, the rates of integration and / or therapeutic efficacy relative to the number of cells administered remain very low, thus necessitating repeated treatment and resulting in very high treatment costs. Furthermore, these low rates and high costs limit the range of diseases and / or patients that can be treated with these methods.

[0011] There are compounds in the state of the art that can improve the tissue environment. For example, agents of

[0012] REPLACEMENT SHEET (RULE 26) Tissue regeneration or RGTA® are known ([1], US8790631). These compounds are known in particular for their use alone or in combination, for example with Hyaluronic Acid ([2], W02020151900), for example in the treatment of tissue damage, such as skin lesions. Tissue Regeneration Agents (TRAs) are known to be useful in treating tissue lesions of various natures and etiologies, as well as from multiple origins, in both humans and veterinary medicine, and in animal models, such as equine, ex vivo, and in vitro ([3], Barritault, et al., 10.1007 / s10719-016-9744-5, 4], Barritault, et al., 10.1016 / j.jbspin.2O16.06.012). The use of Tissue Regeneration Agents (TRAs) with exogenous progenitor cells, such as stem cells of various origins like Mesenchymal Stem Cells (MSCs), is also known ([5], EP3302523B1).

[0013] It is also known that RGTA® may be useful in the treatment of tissue lesions via a replacement of endogenous heparan sulfates destroyed only at the site of the lesion ([6], Meddahi, et al., 10.1002 / jbm.10283), ([7], Pereira, et al., 10.1007 / s10719-022-10047-x) However, after administration, the yields regarding cell integration and / or targeting of the lesion to be treated remain low and are likely to involve possible repetition of treatment and also significant treatment costs. In addition, yields and costs are likely to limit the pathologies and / or patients that can be treated by these processes ([3], Barritault, et al., 10.1007 / s10719-016-9744-5, 4], Barritault, et al., 10.1016 / j.jbspin.2O16.06.012).

[0014] Therefore, there is a real need in the state of the art to find a compound and / or composition that can improve the treatment of tissue lesions.

[0015] The use of Extracellular Vesicles (EVs) for the treatment of tissue damage is also known, with extracellular vesicles being, compared to cells, prepared and stored in large quantities for use on the greatest number of patients ([8], Xin, et al.,

[0016] REPLACEMENT SHEET (RULE 26) 10.1038 / jcbfm.2013.152) ([9], Doeppner, et al., 10.5966 / sctm.2015-0078) (

[0010] , Jeyaram, et al., 10.1208 / s12248-017-0160-y) (

[0011] , Ma, et al., 10.4252 / wjsc.v12.18.814).

[0017] Extracellular vesicles are secreted nanoparticles containing a lipid membrane and bound to phospholipids that transport a complement of lipids, DNA, RNA, proteins, metabolites, and glycans that reflect the identity and molecular state of their cell of origin. The surface chemistry of extracellular vesicles is of great biological importance for regulating the orientation, targeting, and uptake of extracellular vesicles by recipient cells (

[0012] , Yanez-Mo, et al., 10.3402 / jev.v4.27066) (

[0013] , Hallal, et al.,

[0018] 10.1002 / jev2.12260), (

[0014] , Buzas, et al., 10.1007 / s00281-018-0682-0).

[0019] However, after administration, the yields regarding the integration of extracellular vesicles and / or the targeting of the lesion to be treated also remain low and are likely to imply possible repetition of administrations and significant treatment costs. Furthermore, the yields and costs are likely to limit the pathologies and / or patients that can be treated by these methods (

[0015] , Kalimuthu, et al., 10.1186 / sl 3036-019-0160-9), (

[0016] , Uddin, et al., 10.1016 / j.ipha.2024.02.004).

[0020] Therefore, there is a real need in the state of the art to find a compound and / or composition that can improve the treatment of tissue lesions.

[0021] Known treatments for tissue damage may have relative effectiveness.

[0022] Therefore, there is a real need in the state of the art to find a compound and / or composition that improves the effectiveness and / or reduces the costs associated with the treatment of tissue lesions.

[0023] Description of the invention

[0024] The present invention is specifically designed to meet these needs by providing a pharmaceutical composition comprising

[0025] - a biocompatible polymer of the following general formula (I)

[0026] REPLACEMENT SHEET (RULE 26) AaXxYy (I) in which:

[0027] A represents a monomer.

[0028] X represents a grouping R1COOR2 OR -Rg(C=0)Rio,

[0029] Y represents an O or N-sulfonate group corresponding to one of the following formulas -R3OSO3R4, -R5NSO3R6, -R7SO3R8 or R7SO3R8 in which:

[0030] Ri, R3, Rs, and R9 independently represent an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, and which may contain one or more aromatic rings, with the exception of benzylamine and benzylamine sulfonate. R2, R4, Re, and Rs independently represent a hydrogen atom or a cation M + ,

[0031] R7 and R10 independently represent a bond, an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, a represents the number of monomers, x represents the degree of substitution of monomers A by groups X, y represents the degree of substitution of monomers A by groups Y, and

[0032] - at least one extracellular vesicle.

[0033] Advantageously, the inventors have demonstrated in a surprising way that the composition comprising a biocompatible polymer and at least one extracellular vesicle according to the invention advantageously allows the treatment of tissue lesions.

[0034] Advantageously, the inventors have demonstrated in a surprising way that biocompatible polymers according to the invention, for example RGTA®, can advantageously target lesion sites, for example tissue lesions, also referred to as lesion sites, and thus allow at least one extracellular vesicle or extracellular vesicles to be

[0035] REPLACEMENT SHEET (RULE 26) advantageously addressed and sequestered at the site of the lesion to be treated or repaired. Advantageously, the inventors have demonstrated that targeting and / or addressing and / or sequestration at the level of the tissue lesion allows for better treatment of the lesion, for example, accelerated healing and / or tissue regeneration while restoring tissue function. In other words, the composition according to the invention advantageously allows for accelerated healing and / or tissue regeneration and restoration of tissue functionality.

[0036] Furthermore, the inventors have demonstrated, in a surprising and unexpected manner, that the composition comprising a biocompatible polymer and at least one extracellular vesicle according to the invention advantageously enables rapid and functional tissue repair. In particular, the inventors are the first to have demonstrated, in a surprising manner, that after administration, the composition comprising a biocompatible polymer and at least one extracellular vesicle according to the invention is specifically targeted, notably via the biocompatible polymer, to the site of the tissue injury. In other words, the composition according to the invention is specifically targeted to the site of the tissue injury, advantageously accelerating tissue repair and / or promoting reconstruction of the injury site. The composition according to the invention also advantageously and surprisingly enables rapid and optimal treatment of the injury.

[0037] The inventors have also demonstrated that the composition according to the invention advantageously and surprisingly increases cell recruitment to the site of injury. The composition according to the invention, in particular through increased cell recruitment to the site of injury, accelerates tissue repair and / or promotes reconstruction of the lesion site. The composition comprising a biocompatible polymer and at least one extracellular vesicle according to the invention advantageously achieves the same therapeutic effect regardless of the site and / or route of administration. For example, the composition comprising a biocompatible polymer and at least one vesicle

[0038] REPLACEMENT SHEET (RULE 26) extracellular according to the invention can be administered at a distance as well as near the lesion site without modification of its effects concerning the treatment of the lesion and tissue regeneration.

[0039] In other words, the inventors have also demonstrated that the composition according to the invention, after administration, targets the tissue lesion and advantageously allows the treatment of said lesion by recruitment and differentiation of the cells present at the level of the lesion allowing healing of the lesion.

[0040] The inventors have also demonstrated that the composition according to the invention advantageously allows for restoration of the cellular and matrix microenvironment, in particular of the protein and glycan elements of the extracellular matrix at the level of the lesion, notably via stimulation of neosynthesis and remodeling of the extracellular matrix at the level of the lesion.

[0041] The inventors are the first to have demonstrated that the biocompatible polymers according to the invention bind to extracellular vesicles, for example via peptides and / or cell adhesion proteins, transmembrane peptides and / or proteins, extramembrane peptides and / or proteins, secreted peptides and / or proteins, and / or membrane lipoproteins, natural or synthetic, for example synthetic peptides and / or proteins, for example as described in (

[0017] , Fields, 10.1002 / 0471140864. ps1801s26), with a very strong affinity.

[0042] The inventors have also demonstrated in a surprising and unexpected way that the combination of biocompatible polymers and extracellular vesicles, for example the attachment of biocompatible polymers to extracellular vesicles, surprisingly enables the targeting of extracellular vesicles to the tissue injury and / or lesion site.

[0043] The inventors have also demonstrated, in a surprising and unexpected manner, that the combination of biocompatible polymers and extracellular vesicles according to the invention advantageously and surprisingly allows for anchoring of the combination of biocompatible polymers and

[0044] REPLACEMENT SHEET (RULE 26) of extracellular vesicles in the matrix of the damaged tissue. In addition, the inventors have demonstrated, surprisingly, that anchoring can advantageously stimulate locally the biological mechanisms allowing for the reconstruction of the damaged tissue.

[0045] In this context, a monomer is understood, for example, to be a monomer chosen from the group comprising sugars, esters, alcohols, amino acids or nucleotides or derivatives thereof.

[0046] In the present invention, the monomers A constitute the basic elements of the polymers of formula I may be identical or different.

[0047] In the present invention, identical or different monomers A can be independently chosen from sugars or derivatives thereof.

[0048] In the present invention, the monomers A can be independently of the monomers of the following formula: in which Rn and R12 independently represent an oxygen atom, an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, a heteroaryl group independently comprising one or more oxygen and / or nitrogen atoms, an aldehyde function, a carboxylic acid group, a diol, a substituted diol, a group of formula -R13- (X)n-Ru in which R13 represents an aliphatic carbon chain in C1 to C4, possibly branched and / or unsaturated, X represents a heteroatom chosen from oxygen and nitrogen, is an integer from 1 to 4 and R14 is a hydrogen atom, an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, a heteroaryl group independently comprising one or more oxygen and / or nitrogen atoms, an aldehyde function, a carboxylic acid group, a diol, a substituted diol.

[0049] REPLACEMENT SHEET (RULE 26) In the present invention, the association of monomers can make it possible to form a polymeric skeleton, for example a polymeric skeleton of polyester, polyalcohol, polysaccharide nature, of the type of nucleic acids or proteins.

[0050] In the present invention, among the polyesters, it may be for example copolymers of biosynthesis or chemical synthesis, for example aliphatic polyesters or of natural origin for example polyhydroxyalkonates.

[0051] In the present invention, the polysaccharides and their derivatives may be of bacterial, fungal, animal and / or plant origin. They may be, for example, single-chain polysaccharides, e.g. polyglucoses, e.g. dextran, cellulose, beta glucan, starch / glycogen or other monomers comprising more complex units, e.g. xanthan gums, e.g. glucose, mannose, galactose, uronic acids and glucuronic acid, or glucuronans, and glucoglucuronan, galactosamines, iduronic acid and highly glycosylated protein derivatives, e.g. mucins.

[0052] In the present invention, the plant-derived polysaccharides can be single-chain, for example cellulose (glucose), hemicellulose, pectins (galacturonic acid), fucans, starch or more complex such as alginates (galuronic and mannuronic acid).

[0053] In the present invention, the polysaccharides of fungal origin may be, for example, steroglucan.

[0054] In the present invention, the polysaccharides of animal origin may be, for example, chitins or chitosan (glucosamine).

[0055] In the present invention, the monomers A constituting the basic elements of the polymers of formula I can advantageously be identical.

[0056] In the present invention, the monomers A constituting the basic elements of the polymers of formula I can advantageously be glucose.

[0057] REPLACEMENT SHEET (RULE 26) The number of monomers A defined in formula (I) by "a" may be such that the mass of said polymers of formula (I) is greater than or equal to 1000 daltons, for example, greater than 2000 daltons. The number of monomers A defined in formula (I) by "a" may be such that the mass of said polymers of formula (I) is approximately between 1000 and 6000 daltons, for example, which corresponds to at least 5 glucose monomers. For example, the mass of said polymers of formula (I) may be approximately between 3000 and 6000 daltons, for example, which corresponds to 12 to 20 glucose monomers.

[0058] The number of monomers A defined in formula (I) by "a" may also be such that the mass of said polymers of formula (I) is less than about 2,500,000 daltons, for example, which corresponds to 7,000 glucose monomers.

[0059] Advantageously, the mass of said polymers of formula (I) can be from 3000 to 250,000 daltons, for example from 3000 to 6000 daltons, or for example from 20,000 to 250,000 daltons, or for example from 75,000 to 150,000 daltons.

[0060] In the present invention, in the group -R1COOR2 representing X, Ri can be a Ci to Ce alkyl, for example a methyl, an ethyl, a butyl, a propyl, a pentyl, preferably a methyl group, and R2 can be a bond, a Ci to Ce alkyl, for example a methyl, an ethyl, a butyl, a propyl, a pentyl, an R21R22 group in which R21 is an anion and R22 a cation chosen from the alkali metal group.

[0061] Preferably, group X is the group of formula -R1COOR2 in which Ri is a methyl group -CH2- and R2 is an R21R22 group in which R21 is an anion and R22 is a cation chosen from the alkali metal group, preferably group X is a group of formula -CH2-COO- or carboxymethyl.

[0062] In the present invention, in the group -R9(C=0)RIO representing X, R9 can be an alkyl group in C1 to C2, for example a methyl, an ethyl, a butyl, a propyl, a pentyl, preferably a methyl group, and

[0063] REPLACEMENT SHEET (RULE 26) R can be a bond, an alkyl in Ci to Ce, for example a methyl, an ethyl, a butyl, a propyl, a pentyl, a hexyl.

[0064] The rate of substitution of all monomers A by the groups X defined in the general formula (I) by "x" can be from 10 to 150%, from 40 to 80%, and preferably in the order of 50% or 60%.

[0065] In the present invention, in the group corresponding to one of the following formulas -R3OSO3R4, -R5NSO3R6, -R7SO3R8 or R7SO3R8 and representing group Y, R3 may be a bond, an alkyl group in the C1 to C1 ion, for example a methyl, an ethyl, a butyl, a propyl, a pentyl, preferably a methyl group; Rs may be a bond, an alkyl group in the C1 to C1 ion, for example a methyl, an ethyl, a butyl, a propyl, a pentyl, preferably a methyl group; R7 may be a bond, an alkyl group in the C1 to C1 ion, for example a methyl, an ethyl, a butyl, a propyl, a pentyl, preferably a methyl group; R4, Re and Rs may independently be a hydrogen atom or a cation M + , for example M + It could be an alkali metal.

[0066] Preferably, group Y is the group with the formula R7SO3R8, in which R7 is a bond and Rs is an alkali metal selected from the group comprising lithium, sodium, potassium, rubidium, and cesium. Preferably, group Y is a -SO3, -SO3 Na group. +

[0067] The rate of substitution of all monomers A by groups Y defined in the general formula (I) by "y" can be from 10 to 170%, from 30 to 150%, from 55 to 160%, from 55 to 85%, from 120 to 160%, and preferably in the order of 70, 140 or 150%.

[0068] In the present invention, the above definition of substitution rates means that a substitution rate "x" of 100% means that each monomer A of the polymer of the invention statistically contains one group X. Similarly, a substitution rate "y" of 100% means that each monomer of the polymer of the invention statistically contains one group Y. Substitution rates greater than 100% indicate that each monomer statistically carries more than one group of the type considered; conversely, substitution rates less than

[0069] REPLACEMENT SHEET (RULE 26) 100% reflects the fact that each monomer statistically carries less than one group of the type considered.

[0070] Polymers can also include functional chemical groups, designated Z, different from X and Y.

[0071] In the present invention, the Z groups may be identical or different, and may independently be selected from the group comprising amino acids, fatty acids, fatty alcohols, ceramides, or derivatives thereof, or nucleotide targeting sequences, antibodies, antibody fragments.

[0072] The Z groups can also represent identical or different active agents. These may include, for example, therapeutic agents, diagnostic agents, an anti-inflammatory, an antimicrobial, an antibiotic, an antiviral agent, a growth factor, a cell-communication cytokine (e.g., interferon), an enzyme, an antioxidant compound, polyphenols, tannins, anthocyanins, lycopenes, terpenoids, and stilbenes (e.g., resveratrol). In the present invention, the Z group may advantageously be a saturated or unsaturated fatty acid, cholesterol, or a derivative thereof. For example, it may be a cholesterol derivative selected from the group comprising cholecalciferol, cortisone, cortisol, sex hormones, and their derivatives.This may be, for example, a fatty acid chosen from the group including acetic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, trans-vaccenic acid, linoleic acid, linolelaidic acid, α-linolenic acid, γ-linolenic acid, dihomo-γ-linolenic acid, arachidonic acid, eicosapentaenoic acid, clupanodonic acid or docosahexaenoic acid. It may be an eicosanoid, for example chosen from the group including leukotrienes, prostanoids, prostaglandins, thromboxanes and prostacyclins. Preferably, the fatty acid is acetic acid.

[0073] REPLACEMENT SHEET (RULE 26) In the present invention, group Z may advantageously be an amino acid from the L or D series chosen from the group comprising alanine, asparagine, an aromatic chain for example tyrosine, phenylalanine, tryptophan. Preferably, the amino acid is phenylalanine.

[0074] In the present invention, the Z group may be an antioxidant, for example vitamin A, C, E, B9, B6, glutathione, selenium, polyphenols, for example catechins, for example green tea, flavonoids, tannins, anthocyanins, for example red fruits, lycopenes, terpenoids and resveratrol.

[0075] In the present invention, the Z group can be anti-aging compounds, for example retinoids, allantoins.

[0076] In the present invention, the Z group can be antibodies, antibody fragments, for example Fab fragments. It can be, for example, antibodies and / or targeting antibody fragments.

[0077] Advantageously, Z groups can impart additional biological or physicochemical properties to polymers. For example, Z groups can increase the solubility or lipophilicity of the polymer, allowing, for instance, improved diffusion or tissue penetration.

[0078] Advantageously, Z groups can impart additional biological or physicochemical properties to the polymers. Thus, the polymers of the invention, for example when the Z group is selected from an antioxidant compound or an anti-aging compound, can advantageously carry these compounds and thereby provide an additional and / or complementary biological effect.

[0079] Polymers in which Z is present may respond to the following formula II: Aa Xx Yy Zz (II) in which A, X, Y, a, x, y are as defined above and z represents the rate of substitution by Z groups.

[0080] In the present invention the rate of substitution by Z groups represented by "z" can be from 1 to 50%, from 10 to 25%, preferably equal to 15, 20 or 25%.

[0081] REPLACEMENT SHEET (RULE 26) The X, Y, and Z groups may be independently attached to monomer A and / or independently attached to each other. When at least one of the X, Y, and Z groups is independently attached to a different X, Y, and Z group, one of said X, Y, or Z groups is attached to monomer A.

[0082] Thus, the Z groups can be covalently attached directly to the A monomers or covalently attached to the X and / or Y groups.

[0083] In the present invention, the Z groups can also be conjugated to polymers of formula AaXxYy by bonds other than covalent bonds, for example by ionic bonds, e.g. via ionic interactions, hydrophilic bonds, or hydrophobic bonds. The polymers of the invention can then constitute a Z-vectorization system.

[0084] In the present invention, the polymer may be, for example, a polymer selected from the group comprising the compounds OTR4120, OTR41201, OTR41202, OTR41203, OTR41205, OTR41210, OTR41301, OTR41302, OTR41303, OTR41305, OTR41310, OTR3131, OTR4131, OTR4132, preferably OTR4132.

[0085] In the present invention, the polymer may be, for example, a polymer selected from the group comprising the compounds OTR41201, OTR41202, OTR41203, OTR41205, OTR41210, OTR4120, OTR4122, OTR4125, OTR41301, OTR41302, OTR41303, OTR41305, OTR41310, OTR3131, OTR4131, OTR4132, OTR4135, OTR415 with the characteristics mentioned in Table 1 below.

[0086] Table 1: List and characteristics of polymers. (*or phenylalanine methyl ester).

[0087] REPLACEMENT SHEET (RULE 26)

[0088] Table 1: Polymers of the Aa Xx Yy (I) and Aa Xx Yy Zz (II) families in which A is glucose (MW 180 Da), X is CarboxyMethyl (MW 58 Da) Y: SO3 (MW 80 Da) and Z is Acetate (MW 43 Da) or phenylalanine (MW 165 Da).

[0089] In the present invention, the composition may comprise a concentration of 0.001 to 30,000 pg / mL by weight of biocompatible polymer, for example, 0.1 to 300 pg / mL by weight of biocompatible polymer, relative to the volume of the composition. For example, the composition may comprise a concentration of 1 to 300 pg / mL, 10 to 300 pg / mL, or 20 to 200 pg / mL, for example, 1 to 10 pg / mL by weight of biocompatible polymer relative to the total volume of the composition.

[0090] In the present invention, the composition can be formulated and / or adapted according to its administration. For example, for parenteral administration, the composition can be administered to deliver a dose

[0091] REPLACEMENT SHEET (RULE 26) of biocompatible polymer comprising 0.01 to 5 mg per kilogram (kg) of body weight (mg / kg), preferably 0.1 to 1.5 mg per kilogram of body weight. For example, the frequency of administration of the composition may be, for example, once daily, every two days, or every three days, for example, 2 to 3 times per week. For example, the frequency of administration of the composition may be, for example, once every 24 hours, every 48 hours, or every 72 hours, for example, 2 to 3 times per week.

[0092] For example, for oral administration the composition can be administered to deliver a dose of biocompatible polymer of 0.1 to 5 mg per kilogram of body weight, preferably 0.01 to 1.5 mg / kg at a frequency of once daily or twice weekly administration.

[0093] For sublingual administration, the dose can be daily or twice weekly and range from 0.5 pg / kg to 100 pg / kg of body weight.

[0094] For example, for intra-arterial administration, the biocompatible polymer may be at a concentration of 0.01 to 400 pg / mL by weight of biocompatible polymer relative to the total volume of the composition, preferably 1 to 20 mL by slow infusion or bolus.

[0095] For example, for oral administration, the biocompatible polymer may be at a concentration of 0.1 to 100 pg / mL by weight of biocompatible polymer relative to the total volume of the composition, preferably 1 to 50 mL, preferably equal to 5 mL.

[0096] For example, for administration via the airborne route, such as a nasal spray, preferably by inhalation or nebulization, the biocompatible polymer may be at a concentration of 0.1 to 100 pg / mL by weight of biocompatible polymer relative to the total volume of the composition, preferably 1 to 20 mL, preferably 5 mL. This could be, for example, a composition, preferably 1 to 20 mL, of an aqueous solution of OTR4120 at 100 pg / mL or OTR4131 at 10 pg / mL placed in a nebulizer capable of delivering 1 to 3 mL per

[0097] REPLACEMENT SHEET (RULE 26) minutes with an administration time included, for example, from 5 to 10 minutes.

[0098] In this document, a peptide is defined as a polymer of amino acids linked together by peptide bonds. Those skilled in the art, by virtue of their general knowledge, know what is meant by a peptide. It may, for example, be a polymer of amino acids linked together by peptide bonds comprising from 2 to 30 amino acids, for example, from 2 to 20 amino acids. In this document, a synthetic peptide is defined as any synthetic peptide known to those skilled in the art. Those skilled in the art, by virtue of their general knowledge, know what is meant by a synthetic peptide. It may, for example, be peptides obtained by chemical synthesis through the condensation reaction of the carboxyl group of one amino acid onto the amino group of another amino acid, for example, as described in (

[0018] , Chan, et al.) and / or (

[0019] , Benoiton, 10.1201 / 9781420027693). This could include, for example, peptides that are not found in nature.This could be, for example, a peptide synthesized by the solid-phase method ("Solid-Phase Peptide-Synthesis Methodology") as described in (

[0017] , Fields, 10.1002 / 0471140864. ps1801 s26).

[0099] In this context, "protein" means any protein known to a person skilled in the art. A person skilled in the art, by virtue of their general knowledge, understands what is meant by "protein." This could refer, for example, to biological macromolecules present in all living cells, corresponding to polymers formed of one or more polypeptide chains. The length of a polypeptide sequence can, for example, comprise 10,000 to 30,000 amino acids, or 10,000 to 20,000 amino acids. Each polypeptide chain is made up of a chain of amino acid residues linked together by peptide bonds, for example, as described in (

[0020] , San lavi Ile, et al.).

[0100] In this document, the term "synthetic protein" means any synthetic protein known to a person skilled in the art. A person skilled in the art, by virtue of their general knowledge, knows what is meant by a synthetic protein.

[0101] A REPLACEMENT SHEET (RULE 26) may include, for example, proteins obtained by chemical synthesis, such as those comprising synthetic and / or non-synthetic peptides. It may also include proteins comprising synthetic and / or non-synthetic peptides that are not found in nature. Finally, it may include proteins obtained by chemical synthesis whose structure, functions, and amino acid sequences are identical to those of natural proteins.

[0102] In the present invention, the term extracellular vesicle (EV) means any extracellular vesicle known to those skilled in the art. It may be any vesicle produced by living organism cells, for example, selected from the group comprising Archaea, Bacteria, Protozoa, Chromista, Plantae, Fungi, and Animalia. It may be, for example, any vesicle produced and / or originating, for example, from a eukaryotic or prokaryotic cell. It may be, for example, a vesicle produced from eukaryotic or prokaryotic cells, said vesicle comprising a lipid bilayer, lacking a functional nucleus, and incapable of replicating. It may be, for example, at least one extracellular vesicle as described in document (

[0021] , Raposo, et al., 10.1083 / jcb).201211138) and / or characterized in the document "Extracellular vesicles, Definition, separation, characterization", Wilfrid Boireau* and Céline Elie-Caille**, Med Sci (Paris), Volume 37, Number 12, December 2021, Extracellular vesicles, Page(s) 1092 - 1100 (

[0022] , Boireau, et al., 10.1051 / medsci / 2021201). It may be, for example, one or more of the elements produced by cells, particularly in in vitro culture, including vesicles or microvesicles, exosomes, apoptotic bodies and microparticles that are found in the environment in which the cells are kept alive. The vesicles within the meaning of the present invention may comprise a heterogeneous population, the elements of which are differentiable in particular according to their size / diameter and their content.

[0103] In this context, an exosome is defined as an extracellular vesicle released and / or secreted by a cell into its environment, with a size ranging from 30 to 120 nm. It could be, for example, a vesicle

[0104] REPLACEMENT SHEET (RULE 26) extracellular material released and / or excreted by a cell into its environment, with a size ranging from 30 to 90 nm. This could be, for example, an extracellular vesicle released and / or excreted by a cell into its environment as described in (

[0023] , Battistelli, et al., 10.3390 / biology9010021). This could be, for example, an extracellular vesicle released and / or excreted by a cell into its environment, for example, during the fusion of the multivesicular body and the plasma membrane, allowing said extracellular vesicle to be released and / or excreted by a cell into its environment.

[0105] In this context, an apoptotic body refers to a specific type of extracellular vesicle (EV), namely the remnants of cells that have undergone apoptosis, or programmed cell death. This could be, for example, an apoptosome, a multiprotein complex formed by the interaction of cytochrome P450 with APAF-1 and procaspase-9 in the cytosol, following the release of cytochrome P450 from the mitochondria. It could also be extracellular vesicles (EVs) released and / or excreted by a dying cell. Finally, it could be an apoptotic body ranging in size from 500 nm to 2 pm in diameter, or from 600 nm to 1 pm. This could be, for example, an apoptotic body as described in (

[0023] , Battistelli and Falcieri, 10.3390 / biology9010021).

[0106] This may be at least one extracellular vesicle of a eukaryotic cell. In this context, an extracellular vesicle of a "eukaryotic cell" means any extracellular vesicle of a eukaryotic cell known to those skilled in the art. This could be, for example, an extracellular vesicle of a mammalian or plant eukaryotic cell, such as an extracellular vesicle of an animal, human, or plant eukaryotic cell. It could be, for example, an extracellular vesicle of a plant cell as described in (

[0024] , Tan, et al., 10.3389 / fphar.2022.1006299). It could be, for example, any extracellular vesicle of a eukaryotic cell from any biological tissue of mammal known to those skilled in the art. This could be, for example, an extracellular vesicle of a eukaryotic cell from connective tissue, muscle tissue, nerve tissue, bone tissue, cartilage and / or the

[0107] REPLACEMENT SHEET (RULE 26) epithelial tissue. This could be, for example, any extracellular vesicle of a eukaryotic cell at any stage of differentiation, for example, an extracellular vesicle of a cell chosen from the group including adult or embryonic eukaryotic cells, adult stem cells, native or induced pluripotent stem cells (iPSCs), multipotent or unipotent cells, and differentiated cells, with the exception of embryonic stem cells. It could be an extracellular vesicle of eukaryotic cord blood cells, bone marrow cells, adipose tissue cells, mesenchymal cells, muscle cells, tissue cells, for example, joint bone tissue and / or muscle tissue.

[0108] This could include, for example, at least one extracellular vesicle of eukaryotic cells chosen from the group comprising adult stem cells, native or induced pluripotent stem cells (iPS), multipotent or unipotent cells, and differentiated cells or any mixture thereof.

[0109] These may include, for example, vesicles from cells selected from pluripotent cells, such as embryonic stem cells or somatic cells induced to pluripotency or induced pluripotent stem cells (iPS for "induced pluripotent cells"), regardless of somatic origin, i.e., cells taken from adults and reprogrammed into pluripotent cells by various processes including, but not limited to, adenoviruses, plasmids, transposons, Sendai viruses, synthetic mRNAs and recombinant proteins, for example as described in the document Takahashi and Yamanaka, "A decade of transcription factor-mediated reprogramming to pluripotency.", Nat Rev Mol Cell Biol. 2016;17:183-93 (

[0025] , Takahashi, et al., 10.1038 / nrm.2016.8).It may consist of at least one extracellular vesicle of embryonic stem cells, said embryonic stem cells not having been produced by a process involving the destruction of a human embryo.

[0110] This could be, for example, at least one extracellular vesicle of eukaryotic cells, excluding embryonic stem cells.

[0111] REPLACEMENT SHEET (RULE 26) This may include, for example, at least one extracellular vesicle of adult stem cells, for example, at least one extracellular vesicle of adult stem cells from connective tissue, muscle tissue, nerve tissue, adipose tissue, bone tissue, cartilage and / or epithelial tissue.

[0112] This could include, for example, at least one extracellular vesicle of adult eukaryotic cells. It could include, for example, at least one extracellular vesicle of embryonic eukaryotic cells. It could include, for example, extracellular vesicles of embryonic eukaryotic cells from connective tissue, muscle tissue, nervous tissue, adipose tissue, placental tissue, bone, cartilage, and / or epithelial tissue. It could include at least one extracellular vesicle of embryonic eukaryotic cells, provided that these embryonic eukaryotic cells did not originate from a process involving the destruction of a human embryo.

[0113] This could include, for example, at least one extracellular vesicle of native pluripotent or multipotent cells. Examples include extracellular vesicles of native pluripotent or multipotent cells, such as mesenchymal stromal cells or bone marrow stem cells.

[0114] This could be, for example, at least one extracellular vesicle of induced pluripotent stem cells (iPSCs). This could be, for example, an extracellular vesicle of induced pluripotent stem cells (iPSCs) such as those described in Maury Y, Gauthier M, Peschanski M, Martinat C, "Human pluripotent stem cells: opening key for pathological modeling," Med Sci (Paris). 2011 Apr;27(4):443-6. French. (

[0026] , Maury, et al., 10.1051 / medsci / 2011274023).

[0115] This could be, for example, at least one extracellular vesicle of multipotent cells. It could be, for example, an extracellular vesicle of mesenchymal stem cells, such as mesenchymal stem cells from adipose tissue, bone marrow, organ support tissues, bone tissue, cartilage tissue, or other tissues.

[0116] REPLACEMENT SHEET (RULE 26) muscle, bone marrow. These may be, for example, vesicles from cells selected from multipotent cells, for example mesenchymal stem cells, or their differentiated derivatives of ectodermal, endodermal and / or mesodermal origin, for example cells selected from cardiac, vascular, muscular, retinal, adipocytic, tendon, mesothelial, fibrillar, sex, synovial, cutaneous, neural, medullary, osteo-cartilaginous, hepatic, renal, intestinal, hematopoietic, and immune system cells.

[0117] This could be, for example, at least one extracellular vesicle of unipotent cells. It could be, for example, an extracellular vesicle of a hepatocyte, keratinocyte, or myoblast.

[0118] This could be, for example, at least one extracellular vesicle of differentiated cells. It could be, for example, an extracellular vesicle of erythrocytes, corneocytes, myocytes, or primary pneumocytes.

[0119] This may include, for example, at least one extracellular vesicle containing at least one heparan sulfate-binding protein selected from the group comprising prmtl target chromatin (Chtop), tissue factor pathway inhibitor (Tfpi), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (Adamts4), cysteine-rich angiogenic protein 61 (Ccn1), and neuron-derived neurotrophic factor (Ndnf).It may consist of at least one extracellular vesicle from a cell, said extracellular vesicle comprising at least one peptide and / or protein, cell adhesion proteins, transmembrane proteins, secreted proteins, extracellular proteins, intracellular proteins, and / or membrane lipoproteins whose amino acid sequence includes one or more heparan sulfate binding sites. It may consist of at least one extracellular vesicle from a cell, said vesicle comprising a synthetic peptide and / or protein.

[0120] REPLACEMENT SHEET (RULE 26) synthetic comprising one or more binding sites for heparan sulfates. This may be, for example, an extracellular vesicle from a cell comprising at least one peptide, protein, cell adhesion protein, transmembrane protein, secreted protein, extramembrane protein, intracellular protein, and / or membrane lipoprotein with a peptide sequence comprising at least 3 amino acids selected from the group comprising arginine (R), lysine (L) and histidine (H). This may be, for example, an extracellular vesicle of a cell comprising at least one peptide, one protein, one cell adhesion protein, one transmembrane protein, one secreted protein, one extramembrane protein, one intracellular protein, and / or one membrane lipoprotein with a peptide sequence comprising at least 3 amino acids chosen from the group including arginine (R),lysine (L) and histidine (H), said at least peptide, protein, cell adhesion protein, transmembrane protein, secreted protein, extramembrane protein, intracellular protein, and / or lipoprotein having a primary, secondary, tertiary and / or quaternary structure comprising an accumulation and / or concentration of amino acids selected from the group comprising arginine (R), lysine (L) and histidine (H), said at least peptide, protein, cell adhesion protein, transmembrane protein, secreted protein, extramembrane protein, intracellular protein, and / or lipoprotein having a positive charge at a pH between 4.8 and 8.0, for example between 7.3 and 7.5.

[0121] This could be, for example, an extracellular vesicle from a cell comprising at least one peptide, one protein, one cell adhesion protein, one transmembrane protein, one secreted protein, one extramembrane protein, one intracellular protein, and / or one membrane lipoprotein containing a heparan sulfate binding site. The heparan sulfate binding site could be, for example, as described in the document "Importance of specific amino acids in protein binding sites for heparin and heparan sulfate" EE Caldwell 1, VD Nadkarni, JR Fromm, RJ Linhardt, JM Weiler PMID: 8729007 DOI: 10.1016 / 1357-2725(95)00123-9, Int

[0122] REPLACEMENT SHEET (RULE 26) J Biochem Cell Biol. 1996 Feb;28(2):203-16. doi: 10.1016 / 1357-2725(95)00123-9 (

[0027] , Caldwell, et al., 10.1016 / 1357-2725(95)00123-9) and / or as described by Hileman Ronald E, Fromm Jonathan R, Weiler John M, Linhardt Robert J in "Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins" in 1998 in BioEssays (

[0028] , Hileman, et al., 10.1002 / (SICI)1521 - 1878(199802)20:2<156::AID-BIES8>3.0.CO;2-R). The heparan sulfate binding site may be, for example, a peptide of sequence BBXaB or BXaXaBBXaB in which B is any basic amino acid and Xa any amino acid, for example as described in Wu, HF, et al. (1995) Blood 85, 421-428 (

[0029] , Wu, et al., 10.1182 / blood.V85.2.421.421).The heparan sulfate binding site can be, for example, a peptide of sequence XaBBBXaXaBBBXaXaBBXa in which B is any basic amino acid and Xa any amino acid, for example as described in Andersson, E., et al. (2004) Eur J Biochem 271, 1219-1226 (

[0030] , Andersson, et al., 10.1111 / j.1432-1033.2004.04035.x); Sobel, M., et al. (1992) The Journal of biological chemistry 267, 8857-8862 (

[0031] , Sobel, et al., 10.1016 / S0021 - 9258(19)50359-3). The heparan sulfate binding site can be, for example, a peptide with the sequence TXaXaBXaXaTBXaXaXaTBB in which B is any basic amino acid, Xa is any amino acid, and T defines a turn / loop, for example as described in Capila, I. and Linhardt, RJ (2002) Angewandte Chemie International Edition 41, 391-412 (

[0032] , Capila, et al., 10.1002 / 1521-3773(20020201)41:3<390::AID-ANIE390>3.0.CO;2-B); Hileman, RE, et al. (1998) BioEssays 20, 156-167 (

[0028] , Hileman, et al., 10.1002 / (SICI)1521-1878(199802)20:2<156::AID-BIES8>3.0.CO;2-R); Andersson, E., et al. (2004) Eur J Biochem 271, 1219-1226 (

[0030] , Andersson, et al., 10.1111 / j.1432-1033.2004.04035.x).

[0123] This could be, for example, a peptide, a protein, a cell adhesion protein, a transmembrane protein, a secreted protein, an extramembrane protein, an intracellular protein, a membrane lipoprotein comprising at least two, at least three basic amino acids chosen from the group including arginine (R), lysine (L),

[0124] REPLACEMENT SHEET (RULE 26) histidine (H). It may be, for example, a peptide, a protein, a cell adhesion protein, a transmembrane protein, a secreted protein, an extramembrane protein, an intracellular protein, a membrane lipoprotein comprising an amino acid sequence chosen from the group comprising XaXbXcXdXeXf, XhXiXjXkXIXmXnXp and XqXrXsXtXuXvXwXyXzXiX2 in which, Xa, Xd, Xf, Xh, XI, Xm, Xp, Xq, Xr, Xt, Xu, Xw, Xy and Xz are any amino acid and Xb Xc, Xe, Xi, Xj, Xk, Xn, Xs, Xv, Xi and X2 are any basic amino acid.

[0125] In the present, Xa, Xd, Xf, Xh, XI, Xm, Xp, Xq, Xr, Xt, Xu, Xw, Xy and / or Xz can independently be an amino acid of the L or D series.

[0126] This may be, for example, a peptide, a protein, a cell adhesion protein, a transmembrane protein, a secreted protein, an extramembrane protein, an intracellular protein, a membrane lipoprotein comprising an amino acid sequence selected from the group including RLRARM (SEQ ID NO 28), LRKRLLRD (SEQ ID NO 29), PRRARV (SEQ ID NO 30) and CRLYRK (SEQ ID NO 31).

[0127] In the present case, when the peptide, protein, cell adhesion protein, transmembrane protein, secreted protein, extramembrane protein, intracellular protein, or membrane lipoprotein comprising the amino acid sequence XqXrXsXtXuXvXwXxXyXzXiX2, the secondary, tertiary, and / or quaternary structure of the peptide, protein, cell adhesion protein, transmembrane protein, secreted protein, extramembrane protein, intracellular protein, or membrane lipoprotein may independently comprise, prior to the amino acid Xq and / or between the amino acids Xu and Xv and / or between the amino acids Xz and Xi, a turn and / or a T-loop (“turn”).

[0128] This could be, for example, a cell adhesion protein chosen from the group including collagens, fibronectins and / or elastins.

[0129] This could be, for example, a transmembrane protein chosen from the group including tetraspanins, for example CD9 and / or CD81.

[0130] REPLACEMENT SHEET (RULE 26) This may be, for example, an extra-membrane protein chosen from the group comprising membrane receptors, for example chosen from the group comprising fibroblast growth factor receptors (FGFr), secreted proteins, for example growth factors, coagulation cascade proteins and cytokines, for example fibroblast growth factors (FGF), proteins chosen from the group comprising vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFP), brain-derived neurotrophic factor (BDNF), CXCR / SDF chemokine receptor, tissue plasminogen activator (TPA), antithrombin, antithrombin III, thrombin, C-reactive protein, protein C inhibitor, intracellular proteins, for example superoxide dismutase enzymes or SOD.This could be, for example, a membrane lipoprotein, for example chosen from the group including apolipoproteins (APO), Apolipoprotein E (APOE), lipoproteins of different densities, for example low-density lipoprotein, very low-density lipoproteins (VLDL), high-density lipoproteins (HDL).

[0131] In this context, an acidic amino acid means any acidic amino acid known to a person skilled in the art. This could be, for example, an acidic amino acid from the L or D series. It could be, for example, an acidic amino acid chosen from the group comprising aspartic acid (D), glutamic acid (E), asparagine (N), glutamine (Q), serine (S) and threonine (T).

[0132] In this context, a basic amino acid is defined as any basic amino acid known to those skilled in the art. This could be, for example, a basic amino acid from the L or D series. It could also be, for example, a basic amino acid chosen from the group comprising arginine (R), lysine (K), and / or histidine (H).

[0133] Advantageously, the amino acids Xa, Xd, Xf, Xh, XI, Xm, and Xp can be independent, for example, when the peptide and / or protein

[0134] REPLACEMENT SHEET (RULE 26) is in tertiary or quaternary form, orients towards the inside and / or outside of said peptide and / or protein.

[0135] This may be, for example, an extracellular vesicle comprising a peptide and / or a protein and / or a synthetic peptide and / or a synthetic protein such as, for example, cell adhesion proteins, transmembrane proteins, extramembrane proteins, secreted proteins, intracellular proteins and / or membrane lipoproteins and comprising a heparan sulfate binding site as described in the document Heparin-Binding Domains in Vascular Biology Eva M. Munoz and Robert J. Linhardt, Arterioscler Thromb Vase Biol. 2004 Sep; 24(9): 1549- 1557. doi: 10.1161 / 01.ATV.0000137189.22999.3f (

[0033] , Munoz, et al., 10.1161 / 01. ATV.0000137189.22999.3f).This could include, for example, at least one extracellular vesicle containing at least one protein selected from the group comprising the Chromatin target of prmtl (CHTOP) (UniProt: Q9Y3Y2), the Tissue Factor Pathway Inhibitor Protein TFPI (UniProt: P10646), Disintegrin and Metalloproteinase with Thrombospondin Motifs 4 (ADAMTS4, UniProt: O75173-ATS4), the cysteine-rich angiogenic protein 61 also known as Ccn1 (CCN1, UniProt: 000622), Human Neuron Derived Neurotrophic Factor (NDNF, UniProt: Q8TB73), and fibronectin (UniProt: P02751-FINC) or collagen 1 (UNiProt: P02452-CO1A1), said at least one protein comprising at least one heparan sulfate binding site.In the present for the identification of proteins, the UniProt Consortium nomenclature is used UniProt: the Universal Protein Knowledgebase in 2023 (

[0034] , UniProt, 10.1093 / nar / gkac1052).

[0136] Advantageously, basic amino acids can be positively charged at physiological pH due to the presence of one or more basic amino acids, namely a pH between 7 and 7.7, preferably 7.4, and can advantageously form a positively charged group / region within the protein or peptide. Advantageously, said

[0137] REPLACEMENT SHEET (RULE 26) Basic amino acids can form a positively charged group / region within a protein or peptide, for example, when it is in quaternary form. This could be, for example, a protein or peptide containing a positively charged group / region within the protein or peptide that is identical or similar to collagen 7 (UniProt: CO7A1-Q02388). This may be, for example, a protein or peptide comprising a peptide sequence including at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight peptide(s) selected from the group including RVRAQHRERVT (SEQ ID NO 1): SVRLRGLRPL (SEQ ID NO 2), ARGYRLEWRR (SEQ ID NO 3), HRAEATRRVLER (SEQ ID NO 4), PGRRQH (SEQ ID NO 5), RRRGPKG (SEQ ID NO 6), RGERGEK (SEQ ID NO 7), RGLKGERGVK (SEQ ID NO 8), RWYHR (SEQ ID NO 9).For example, it could be a protein with a tertiary structure as shown in Figure 7.

[0138] This could be, for example, at least one extracellular vesicle comprising at least one protein chosen from the group comprising transmembrane proteins, for example tetraspanins, for example CD9, CD63, CD81 and / or CD82.

[0139] This could be, for example, at least one extracellular vesicle comprising at least one protein chosen from the group comprising cytosolic proteins, for example Alix and / or TSG101.

[0140] This could be, for example, at least one extracellular vesicle comprising at least one protein chosen from the group comprising annexins.

[0141] This could be, for example, at least one extracellular vesicle containing at least one protein chosen from the group including immunoregulators (MHC).

[0142] This could be, for example, at least one extracellular vesicle containing at least one protein chosen from the group including integrins, for example D2C9

[0143] REPLACEMENT SHEET (RULE 26) This may be, for example, at least one extracellular vesicle comprising at least one protein selected from the group comprising immunoglobulins, for example CD90.

[0144] This could be, for example, at least one extracellular vesicle containing at least one protein chosen from the group including CD73 and CD44.

[0145] This could include, for example, at least one extracellular vesicle extracted and / or isolated from a sample of a biological fluid. This could include, for example, at least one extracellular vesicle extracted and / or isolated from a sample of a biological fluid chosen from the group including blood, urine, saliva, semen, cerebrospinal fluid, serum, tear fluid, amniotic fluid, synovial fluid, milk,

[0146] This could be an extracellular vesicle of a prokaryotic cell. In this context, "extracellular vesicle of a prokaryotic cell" means any extracellular vesicle of a prokaryotic cell known to those skilled in the art. This could include, for example, extracellular vesicles of bacteria.

[0147] The choice of cells, and consequently of vesicles which are used for the implementation of the present invention, is of course dependent on the therapeutic target aimed at when using the composition of the present invention, for example the tissue lesion and / or deficient tissue to be repaired.

[0148] According to the invention, the preparation of vesicles usable for implementing the present invention can be carried out using any suitable method known to those skilled in the art. This could be, for example, an in vitro method comprising culturing said cells, with the vesicles being secreted by the cells in question into their culture medium, which has been previously verified to contain no (or very few) vesicles that would naturally represent a confounding factor. The extracellular vesicles are collected from these conditioned media. The following documents describe cell culture methods and the preparation of vesicles from these cultures usable for implementing the present invention, using different cell types:

[0149] REPLACEMENT SHEET (RULE 26) - (

[0035] , Akbar, et al., 10.3390 / cellsl 1020186)

[0150] - (

[0036] , Clos-Sansalvador, et al., 10.1016 / j.ejcb.2022.151227)

[0036]

[0151] (

[0037] , Liangsupree, et al., 10.1016 / j.chroma.2020.461773)

[0152] - (

[0038] , Brennan, et al., 10.1038 / s41598-020-57497-7).

[0153] It may be an extracellular vesicle further comprising at least one other active ingredient; particularly another therapeutically active ingredient; said active ingredient being internalized and / or adsorbed and / or grafted onto the extracellular vesicles.

[0154] The active ingredient may be, for example, any pharmaceutical product known to those skilled in the art. It may be, for example, an active ingredient used in the treatment of opportunistic infections, such as those caused by a microorganism, such as a virus or bacterium, which may develop in a patient with tissue damage. The active ingredient may be, for example, at least one antibiotic, at least one anti-inflammatory, and at least one antiviral. It may be, for example, an extracellular vesicle further comprising at least one other active ingredient; particularly another therapeutically active ingredient; as described in (

[0039] , Panigrahi, et al., 10.1016 / j.tranon.2022.101439).

[0155] In this document, "pharmaceutical composition" means any form of pharmaceutical composition known to a person skilled in the art.

[0156] The composition according to the invention may include any pharmaceutically acceptable excipient.

[0157] The composition according to the present invention may also include various diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. More generally, it may also include a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable excipient.

[0158] As used here, the term "pharmaceutically acceptable carrier" refers to all solvents, buffers, saline solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption retardants, and other physiologically acceptable substances.

[0159] REPLACEMENT SHEET (RULE 26) compatible. The type of carrier can be chosen according to the intended route of administration. In various embodiments, the vehicle is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal, or oral administration. Pharmaceutically acceptable vehicles include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of media and agents for pharmaceutically active substances is well known in art.

[0160] In this context, the pharmaceutical composition may be, for example, an injectable solution. This could be, for instance, an injectable solution for local or systemic injection, such as physiological saline or glucose injectable solution, with or without excipients, such as Dextrans, at concentrations known to those skilled in the art, such as from micrograms to a few milligrams per mL. The pharmaceutical composition may also be, for example, a medicinal product for oral administration selected from the group comprising a liquid formulation, an effervescent oral dosage form, an oral powder, a multiparticle system, or an orodispersible dosage form.

[0161] For example, when the pharmaceutical composition is for oral administration, it may be in the form of a liquid formulation chosen from the group including a solution, syrup, suspension, or emulsion. When the pharmaceutical composition is in the form of an effervescent oral dosage form, it may be in a form chosen from the group including tablets, granules, or powders. When the pharmaceutical composition is in the form of an oral powder or a multiparticle system, it may be in a form chosen from the group including beads, granules, mini-tablets, and microgranules. When the pharmaceutical composition is in the form of an orodispersible dosage form, it may be in a form chosen from the group including orodispersible tablets and wafers.

[0162] REPLACEMENT SHEET (RULE 26) freeze-dried, thin films, a chewable tablet, a tablet, a capsule or a medicated chewable gum.

[0163] According to the present invention, the pharmaceutical composition can be a pharmaceutical composition for administration by oral route, for example buccal and / or sublingual, for example selected from the group including buccal or sublingual tablets, lozenges, drops, a solution for sprays.

[0164] According to the present invention, the pharmaceutical composition can be a pharmaceutical composition for topical, transdermal administration, for example chosen from the group including ointments, creams, gels, lotions, patches and foams.

[0165] According to the present invention, the pharmaceutical composition can be a pharmaceutical composition for administration by respiratory or nasal route, for example in the form of an aerosol, for example administered with a nebulizer and / or inhaler.

[0166] According to the present invention, the composition may be a composition for nasal or respiratory administration, for example selected from the group comprising nasal drops, nasal spray, nasal powder, aerosols, for example compressed gas aerosols and / or nasal spray, or nebulizers

[0167] According to the present invention, the pharmaceutical composition can be a pharmaceutical composition suitable for intrapulmonary administration.

[0168] According to the present invention, the pharmaceutical composition can be a pharmaceutical composition for parenteral administration, for example subcutaneous, intramuscular, intravenous, intra-arterial, intracranial, intrathecal.

[0169] According to the present invention, the pharmaceutical composition can be a pharmaceutical composition for administration via the ocular route, for example selected from the group comprising eye drops, ophthalmic ointment, ophthalmic gel, eye bath.

[0170] REPLACEMENT SHEET (RULE 26) The composition of the present invention may also include at least one other active ingredient, particularly another therapeutically active ingredient, for example, for simultaneous, separate, or staggered use depending on the pharmaceutical formulation used. This other ingredient may, for example, be an active ingredient used in the treatment of opportunistic infections that may develop in a patient with tissue damage caused by a microorganism, for example, a virus or bacteria. It may also be a pharmaceutical product known to those skilled in the art, for example, antibiotics, anti-inflammatories, antivirals, or anticancer agents.

[0171] The composition of the present invention may also include at least one other active ingredient, particularly another therapeutically active ingredient, for example, for simultaneous, separate, or staggered use depending on the pharmaceutical formulation used. This other ingredient may be, for example, an active ingredient used, for instance, in the treatment of opportunistic diseases, or a vitamin, for example, vitamin C used at a high dose, for example, 50 to 100 mg / kg / day, or an analgesic, or an antibiotic, or a bronchodilator, for example, salbutamol, or a corticosteroid, for example, methylprednisolone, or an antiviral, for example, interferon alfa-2b or lopinavir.

[0172] The composition of the present invention may also further comprise hyaluronic acid.

[0173] In the present case, the administration of the biocompatible polymer and hyaluronic acid may be simultaneous, successive, or concomitant.

[0174] According to the invention, at least one of the administrations can be carried out topically, orally, by inhalation, or by injection, preferably by inhalation. The two administrations can be carried out in the same way or differently. For example, the biocompatible polymer and hyaluronic acid can be administered by inhalation. The administration method can also be determined by the area and / or biological tissue to be treated.

[0175] REPLACEMENT SHEET (RULE 26) According to the invention, the composition can, for example, be administered only once.

[0176] According to the invention, the composition can also be administered, for example, daily, twice daily, weekly, or less frequently. This could include, for example, administration once a day, twice a day, or less frequently, such as once every two days, or weekly.

[0177] According to the invention, and depending on the method of administration, the composition can be, for example, a saline composition administered daily, twice daily, weekly, or less frequently. It could be, for example, administered once a day, twice a day, or less frequently.

[0178] According to the invention, the composition can be administered, for example, over a period of 1 day to 3 months, for example for 2 months, for example for 1 month, for example for one week. For example, the composition can be administered over a period of 1 to 3 weeks, for example with a frequency of administration every day or every other day.

[0179] For example, when the composition is in a form suitable for administration by inhalation, the composition may preferably be administered with a frequency of administration every two or three days.

[0180] According to the invention, the composition can be administered, for example, daily, twice daily, and weekly. This could be, for example, once a day, twice a day, or more frequently.

[0181] According to the invention, the composition can be administered, for example, over a period of 1 day to 3 months, for example, for 2 months. For example, the composition can be administered over a period of 3 months with a frequency of administration every day.

[0182] The present invention also relates to a pharmaceutical composition comprising at least one biocompatible polymer of general formula (I) or (II) and at least one extracellular vesicle, said at least one biocompatible polymer of general formula (I) or (II) being on the surface of said at least one extracellular vesicle.

[0183] REPLACEMENT SHEET (RULE 26) According to the invention, the biocompatible polymer of formula (I) or (II) is as defined above.

[0184] According to the invention, the extracellular vesicle is as defined above.

[0185] According to the invention, the pharmaceutical composition is as defined above.

[0186] The present invention also relates to a pharmaceutical composition comprising at least one biopolymer of formula AaXxYy or AaXxYyZz and at least one extracellular vesicle as a drug.

[0187] According to the invention, the extracellular vesicle is as defined above.

[0188] According to the invention, the biopolymer of formula AaXxYy or AaXxYyZz is as defined above.

[0189] According to the invention, the pharmaceutical composition is as defined above.

[0190] According to the invention, the frequency of administration of the pharmaceutical composition can be as defined above.

[0191] According to the invention, the method and / or route of administration of the pharmaceutical composition may be as defined above.

[0192] According to the invention, said at least one biocompatible polymer of general formula (I) or (II) may be on the surface of said at least extracellular vesicle.

[0193] The present invention also relates to the pharmaceutical composition comprising a biopolymer of formula AaXxYy or AaXxYyZz and at least one extracellular vesicle for its use in the prevention and / or treatment of tissue damage.

[0194] According to the invention, the extracellular vesicle is as defined above.

[0195] According to the invention, the biopolymer of formula AaXxYy or AaXxYyZz is as defined above.

[0196] REPLACEMENT SHEET (RULE 26) According to the invention, the pharmaceutical composition is as defined above.

[0197] According to the invention, the frequency of administration of the pharmaceutical composition can be as defined above.

[0198] According to the invention, the method and / or route of administration of the pharmaceutical composition may be as defined above.

[0199] According to the invention, said at least one biocompatible polymer of general formula (I) or (II) may be on the surface of said at least one extracellular vesicle.

[0200] In this document, "tissue lesions" means any lesion of any biological tissue of a mammal known to those skilled in the art. This could include, for example, damage to or an abnormal change in the tissue of an organism, such as that caused by injury or disease. It could include any tissue lesion known to those skilled in the art, regardless of the shape and / or size of the lesion. It could include, for example, a tissue lesion with or without inflammation, preferably a tissue lesion without inflammation. It could include, for example, a lesion such as that described in Adolphs, 10.1016 / j. neuron.2016.05.014

[0043] . It could include, for example, a lesion of connective tissue, muscle tissue, nerve tissue, bone, cartilage, and / or epithelial tissue. This could include, for example, any lesion of any organ or organelle of a mammal known to a person skilled in the art.This could include, for example, lesions of the skin, mucous membranes, tissues of the digestive tract, tissues of the gastrointestinal tract, the digestive system (nutrition and excretion), the genital tract, the reproductive system, the optical, olfactory or auditory system, the sensory system, the circulatory and / or cardiovascular system, the respiratory system, the muscular system, the locomotor system, the central nervous system, and the peripheral nervous system. It could also include, for example, lesions of the gastric tissue, oral lesions (e.g., periodontal lesions), inflammatory oral lesions (e.g., mucositis), corneal lesions, tympanic membrane lesions, cochlear lesions, skin lesions (e.g., wounds), chronic wounds, and sores.

[0201] REPLACEMENT SHEET (RULE 26) diabetic, ulcerative wound, pressure ulcer, skin burn, necrotizing wound, venous injury, ischemic injury, e.g. ischemic necrosis, injury due to an infarction, e.g. myocardial infarction, bone injury, e.g. fracture, fracture with bone defect, osteonecrosis (“non-union bone fracture”), osteochondral injury, cartilage injury, tendon injury, surgical injury, injury due to surgery, injury due to medical treatment, e.g. radiotherapy, nerve tissue injury, e.g. brain injury, e.g. injury due to tumor removal, spinal cord injury, nerve fiber injury, e.g. of the locomotor and / or sensory system, e.g. neuromuscular injury, respiratory system injury, e.g. lung injury, injury to the circulatory system,For example, damage to the arteries and / or vessels, damage to the digestive system, kidney damage, liver damage, urinary tract damage, or damage to the tissues of the oral cavity.

[0202] It can also refer to any lesion of any biological tissue of a plant known to a person skilled in the art. For example, it could refer to any lesion of any plant organ or organelle known to a person skilled in the art.

[0203] In this context, an "effective amount" is an amount that is sufficient to treat an injury or produce a stated effect. For example, an effective amount might be one that is effective in reducing the progression or severity of the condition or lesions being treated. Determining a therapeutically effective amount is largely within the capabilities of a person skilled in the art. The term "effective amount" is intended to include an amount of a compound described herein, or an amount of a combination of compounds described herein, that is effective in treating or preventing an injury, or in treating the symptoms of the injury, in a host. Therefore, an "effective amount" generally means an amount that produces the desired effect.

[0204] The terms "treat", "treat" and "treatment" include (i) the prevention of the onset of a disease, pathological or medical condition (e.g., prophylaxis); (ii) the inhibition of the lesion or the cessation of its development;

[0205] REPLACEMENT SHEET (RULE 26) (iii) relief of the injury; and / or (iv) reduction of symptoms associated with the injury. Thus, the terms "treat," "treatment," and "care" extend to prophylaxis and include the prevention, prevention, reduction, cessation, or reversal of the progression or severity of the condition or symptoms being treated, e.g., of the treated injury. Thus, the term "treatment" includes the administration of medical, therapeutic, and / or prophylactic medication, as appropriate.

[0206] The present invention also relates to a method of treating a patient who has suffered tissue damage comprising the administration of a pharmaceutical composition comprising a biopolymer of formula AaXxYy or AaXxYyZz and at least one extracellular vesicle.

[0207] According to the invention, the extracellular vesicle is as defined above.

[0208] According to the invention, the biopolymer of formula AaXxYy or AaXxYyZz is as defined above.

[0209] According to the invention, the pharmaceutical composition is as defined above.

[0210] According to the invention, the frequency of administration of the pharmaceutical composition can be as defined above.

[0211] According to the invention, the method and / or route of administration of the pharmaceutical composition may be as defined above.

[0212] According to the invention, the patient can be any mammal. It could be, for example, an animal or a human being.

[0213] The present invention also relates to a pharmaceutical kit for the prevention and / or treatment of tissue damage comprising: i. a biocompatible polymer, and ii. at least one extracellular vesicle.

[0214] The biocompatible polymer is as defined above.

[0215] The extracellular vesicle is as defined above.

[0216] REPLACEMENT SHEET (RULE 26) The inventors have demonstrated, in a surprising and unexpected manner, that extracellular vesicles express proteins which, when associated with the biocompatible polymer according to the invention, advantageously allow the polymer to bind to said extracellular vesicles with high affinity. Furthermore, the inventors have demonstrated, in a surprising and unexpected manner, that the biocompatible polymers according to the invention can bind, for example, through attractive intermolecular bonds, such as those resulting from dipole-dipole interaction, such as hydrogen bonds, hydrophobic bonds, van der Waals forces, preferably electrostatic bonds, and / or ionic bonds.In particular, the inventors have surprisingly demonstrated that the biocompatible polymers according to the invention can bind to proteins present on the membrane surface of said extracellular vesicles, for example peptides and / or proteins mentioned above, for example extracellular proteins and / or transmembrane proteins for example the Chtop protein (“Chromatin target of prmtl”), the tissue factor pathway inhibitor protein (Tfpi), the protein disintegrin and metalloproteinase with thrombospondin motifs 4 (“Disintegrin And Metalloproteinase with Thrombospondin Motifs 4” (Adamts4)), the cysteine-rich angiogenic protein 61 also designated Ccn1 and / or the Human Neuron Derived Neurotrophic Factor (“Human Neuron Derived Neurotrophic Factor” (Ndnf)).

[0217] The inventors have demonstrated in a surprising and unexpected manner that the combination of extracellular vesicles and a biocompatible polymer according to the invention advantageously protects proteins, for example, chromatin target of prmtl (Chtop), tissue factor pathway inhibitor (Tfpi), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (Adamts4), cysteine-rich angiogenic protein 61 (Ccn1), and neuron-derived neurotrophic factor.

[0218] REPLACEMENT SHEET (RULE 26) (Ndnf)) present on the surface of said extracellular vesicles. In particular, the inventors have demonstrated in a surprising and unexpected manner that the combination of extracellular vesicles and a biocompatible polymer according to the invention advantageously protects proteins present on the surface of said extracellular vesicles from proteolysis.

[0219] The inventors have also demonstrated in a surprising and unexpected way that the biocompatible polymer according to the invention advantageously protects proteins present on the surface of said extracellular vesicles from proteolysis, for example via the attachment of said biocompatible polymer to said proteins.

[0220] The inventors have demonstrated in a surprising way that the combination according to the invention advantageously allows the biocompatible polymer according to the invention to be fixed to the surface of the extracellular vesicle and, moreover, surprisingly allows the said vesicle to be protected from proteolysis.

[0221] The present invention also relates to an extracellular vesicle comprising on its surface at least one biocompatible polymer of general formula (I) or (II).

[0222] The biocompatible polymer of formula (I) or (II) is as defined above.

[0223] The extracellular vesicle is as defined above.

[0224] In this context, the term "extracellular vesicle surface" refers to the surface of the vesicle's outer membrane. This could be, for example, the surface of the vesicle's outer membrane facing the external environment. It could also be, for example, the exofacial coronal layer surrounding the extracellular vesicle membrane. The vesicle membrane facing the external environment and / or the exofacial coronal layer may contain molecules, such as peptides and / or proteins derived from the cells of origin and acquired through electrostatic interactions with extracellular molecules.

[0225] REPLACEMENT SHEET (RULE 26) Advantageously, peptides and / or proteins present on the surface of extracellular vesicles can contribute to molecular properties, for example, to the targeting of extracellular vesicles, for example, to cells, for example, via specific receptors and / or proteins and / or peptides. Advantageously, vesicles targeted to said cells can be internalized into said cells. Advantageously, peptides and / or proteins present on the surface of extracellular vesicles can contribute to physical molecular properties, for example, modifications of membrane fluidity, and / or electrostatic properties, for example, by giving the extracellular vesicle a specific overall charge.This coronal layer regulates intercellular interactions, mobility and biodistribution of extracellular vesicles, and often contributes to multiple key features for the identification, classification and affinity isolation of extracellular vesicles (

[0013] , Hallal, et al., 10.1002 / jev2.12260) (

[0014] , Buzas, et al., 10.1007 / S00281 -018-0682-0).

[0226] Advantageously, the biocompatible polymer of formula (I) or (II) can be adhered to, absorbed, and / or attached to the surface of the extracellular vesicle. For example, the affinity constant (KD) between the extracellular vesicle and the biocompatible polymer of formula (I) or (II) can be from 10⁻¹⁰ 12 at 10' 8 M, for example, of 10' 11 at 10' 8 Mr.

[0227] Advantageously, the extracellular vesicle comprising on its surface at least one biocompatible polymer of general formula (I) or (II) can be addressed to tissue injury sites, for example comprising peptides and / or proteins having binding sites, for example to heparan sulfate as mentioned above.

[0228] The present invention also relates to an extracellular vesicle comprising on its surface at least one biocompatible polymer of general formula (I) or (II) for its use in the prevention and / or treatment of tissue injury.

[0229] The tissue injury is as defined above.

[0230] REPLACEMENT SHEET (RULE 26) The present invention also relates to a method for preparing extracellular vesicles with at least one biocompatible polymer comprising the steps of: a. culturing at least one cell in a medium M1 b. culturing said at least one cell obtained in step a in a medium M2 comprising a biocompatible polymer, c. Retrieving the extracellular vesicles obtained in step b. from the medium M2.

[0231] The biocompatible polymer is as defined above.

[0232] The extracellular vesicle is as defined above.

[0233] In this context, a cell is defined as above. It may be, for example, a mammalian or plant eukaryotic cell. It may be, for example, a eukaryotic cell from any mammalian biological tissue known to those skilled in the art. It may be, for example, a eukaryotic cell from connective tissue, muscle tissue, nervous tissue, bone, cartilage, and / or epithelial tissue. It may be, for example, any eukaryotic cell at any stage of differentiation, for example, a cell chosen from the group comprising adult or embryonic eukaryotic cells, adult stem cells, native or induced pluripotent stem cells (iPSCs), multipotent or unipotent cells, and differentiated cells, with the exception of embryonic stem cells.This could be eukaryotic cells from cord blood, bone marrow, adipose tissue, mesenchymal, muscle, or other tissues, such as joint bone and / or muscle tissue. It could be, for example, a eukaryotic cell selected from the group including adult stem cells, native or induced pluripotent stem cells (iPSCs), multipotent or unipotent cells, and differentiated cells, or any mixture thereof. It could be, for example, an adult stem cell, such as an adult stem cell from connective tissue, muscle tissue, nerve tissue, bone, cartilage, and / or other tissues.

[0234] REPLACEMENT SHEET (RULE 26) epithelial tissue. This could be, for example, an adult eukaryotic cell. This could be, for example, an embryonic eukaryotic cell.

[0235] This could be, for example, an embryonic eukaryotic cell from connective tissue, muscle tissue, nervous tissue, bone, cartilage, and / or epithelial tissue. It could be, for example, a native pluripotent cell. It could be, for example, cells. It could be, for example, an induced pluripotent stem cell (iPSC). It could be, for example, an induced pluripotent stem cell (iPSC) as described in Maury Y, Gauthier M, Peschanski M, Martinat C [Ref 10.1051 / medsci / 2011274023], Human pluripotent stem cells: opening key for pathological modeling, Med Sci (Paris). 2011 Apr;27(4):443-6. French, doi: 10.1051 / medsci / 2011274023. Epub 2011 Apr 28. PMID: 21524412

[0027] It could be, for example, a multipotent cell.This could be, for example, a mesenchymal stem cell, such as a mesenchymal stem cell from adipose tissue, bone marrow, organ support tissues, bone tissue, cartilage tissue, muscle tissue, or bone marrow. It could also be, for example, a unipotent cell, such as a hepatocyte, keratinocyte, or myoblast. Finally, it could be a differentiated cell, such as an erythrocyte, corneocyte, myocyte, chondrocyte, hepatocyte, cholangiocyte, synoviocyte, oocyte, or spermatozoa.

[0236] It could be a prokaryotic cell. For example, it could be any prokaryotic cell known to a person skilled in the art that is capable of producing at least one extracellular vesicle. It could, for example, be a bacterial cell.

[0237] In this document, "M1 medium" refers to any culture medium known to those skilled in the art that is suitable for the culture of eukaryotic, prokaryotic, or plant cells. This could be, for example, a commercially available eukaryotic cell culture medium. It could also be, for example, a eukaryotic cell culture medium selected from the group that includes Dulbecco's Modified Eagle's Medium (DMEM) - low

[0238] REPLACEMENT SHEET (RULE 26) glucose (D4655, Sigma-Aldrich), supplemented with 1% penicillin-streptomycin (P4333, Sigma-Aldrich), 1% L-glutamine (G7513, Sigma-Aldrich), and 10% fetal bovine serum (FB-1001, Biosera). This could be, for example, a commercially available prokaryotic cell culture medium. For example, a commercially available prokaryotic cell culture medium, such as lysogeny broth (LB), for example, LB medium, L2542, marketed by Sigma-Aldrich. This could be, for example, a commercially available plant cell culture medium such as Gamborg B5 medium (G5893, Sigma-Aldrich).

[0239] A person skilled in the art, through their general knowledge, will be able to adapt the culture medium according to the cells used.

[0240] In this text, medium M2 means any medium known to those skilled in the art suitable for cell incubation and the isolation of extracellular vesicles. For example, for eukaryotic cells, this could be Dulbecco's Modified Eagle's Medium (DMEM) - low glucose (D4655, Sigma-Aldrich), supplemented with 1% penicillin-streptomycin (P4333, Sigma-Aldrich) and 2 mM L-glutamine (G7513, Sigma-Aldrich).

[0241] According to the invention, the medium M2 can comprise a biocompatible polymer concentration of 1 picogram ms / mL to 10 mg / mL, preferably from 0.1 to 100 pg / mL

[0242] In the present, the duration of step a) of culture may be from 3 to 5 days, preferably ... days.

[0243] In the present, the cell culture of step a) can be carried out under a controlled atmosphere comprising 5% CO2.

[0244] According to the invention, step a) of cell culture can be carried out at a temperature between 4°C and 45°C, for example at 37°C or 4°C. For example, when the cells are eukaryotic cells, for example mammalian cells, cell culture can be carried out at a temperature of 37°C. For example, when the cells are prokaryotic cells, for example bacteria, fungi, yeast, and plant cells, cell culture can be carried out at a temperature of 4°C.

[0245] REPLACEMENT SHEET (RULE 26) °C. For example, when the cells are heat-resistant bacteria, for example heat-resistant Escherichia coli also referred to as fecal compliant, cell culture can be carried out at a temperature of 43 °C to 45 °C.

[0246] According to the invention, the duration of step b) of culture can be from 20 to 48 hours, for example from 24 hours.

[0247] In the present, the cell culture of step b) can be carried out under a controlled atmosphere at 5% CO2.

[0248] According to the invention, step a) of cell culture can be carried out at a temperature of 30 to 40°C, for example 37°C.

[0249] According to the invention, step a) can be carried out in any suitable culture container known to those skilled in the art. This could be, for example, a 75 cm³ flask. 2 and / or any flask known to those skilled in the art that allows for cell culture. This could include, for example, multi-well plates, such as 6-well plates, 12-well plates, 24-well plates, 48-well plates, and / or 96-well plates. It could also include, for example, a bioreactor, for example, containing a culture medium volume of 50 mL to 100 L.

[0250] According to the invention, step c) of extracellular vesicle recovery can be carried out by any suitable method known to those skilled in the art. This could be, for example, a method comprising a centrifugation step, a filtration step, and ultracentrifugation. For example, step b) of extracellular vesicle recovery could include

[0251] - a step b' of recovery of the medium M2,

[0252] - a centrifugation step b”,

[0253] - a step b'” of recovering the supernatant obtained in step b” and filtering said supernatant, and

[0254] - a step b”” of ultracentrifugation, washing of the pellet including the extracellular vesicles with a solution and removal of said washing solution.

[0255] REPLACEMENT SHEET (RULE 26) In this procedure, in step b', the recovery of the medium may be carried out by any method known to a person skilled in the art. This may be, for example, aspiration of the medium. For example, aspiration of the medium may be carried out by any suitable device known to a person skilled in the art, for example, a sterile pipette, for example, a 10 mL pipette. In this procedure, the aspirated medium may be delivered into any suitable container, for example, a tube, for example, a 50 mL tube.

[0256] In the present case, step b” of centrifugation can be carried out by any method known to those skilled in the art. This could be, for example, centrifugation at 2,000 to 10,000 x g, for example, at 2,000 to 10,000 g, for example, at 10,000 g.

[0257] According to the invention, step b” of centrifugation can be carried out for a period of 5 to 40 min, for example 20 to 30 minutes.

[0258] In this procedure, in step b', the recovery of the supernatant can be carried out by any suitable method known to those skilled in the art. This could be, for example, aspiration of the medium. For example, aspiration of the medium can be carried out using any suitable device known to those skilled in the art, for example, a sterile pipette, for example, a 10 mL pipette. In this procedure, the aspirated medium can be delivered into any suitable container, for example, a tube, for example, a 50 mL tube.

[0259] In the present case, in step b'”, the filtration of the supernatant can be carried out by any method known to a person skilled in the art. This could be, for example, filtration through a membrane, for example, filtration with a membrane having a pore diameter of 0.20 to 0.30 µm, for example, 0.22 µm.

[0260] In the present, in step b, centrifugation can be carried out by any method known to those skilled in the art. This may be centrifugation according to the method described in (

[0040] , Xu, et al., 10.1080 / 08958378.2019.1597220, 41], Thery, et al.,

[0261] 10.1002 / 0471143030. cb0322s30). This could be, for example, centrifugation at a speed of 30,000 to 45,000 g, for example at a speed of 34,000 revolutions per minute (100,000 x g). In step b, the centrifugation

[0262] REPLACEMENT SHEET (RULE 26) can be completed for a period of 5 to 80 min, for example 65 to 75 minutes, for example 70 minutes.

[0263] In the present, in step “b”, washing of the pellet including the extracellular vesicles can be carried out by any method known to those skilled in the art, for example, by spraying, soaking of the pellet in a washing solution.

[0264] In this document, "washing solution" means any extracellular vesicle washing solution known to those skilled in the art. This could be, for example, phosphate-buffered saline (PBS) with a pH between 7 and 7.8, for example 7.4. It could also be a commercially available buffer solution, for example, a 1X phosphate-buffered saline (PBS) solution marketed by Eurobio.

[0265] In the present case, in step b, the removal of the pellet washing solution can be carried out by any suitable method known to those skilled in the art. This could be, for example, centrifugation, for example, ultracentrifugation, at a speed of 30,000 to 45,000 rpm, for example, at a speed of 34,000 rpm (100,000 x g) for a duration of 5 to 80 min, for example, 65 to 75 min, for example, for 70 min. Advantageously, this step pellets the washed extracellular vesicles. The washing solution can then be removed by any suitable method known, for example, by aspiration, for example, with a sterile pipette.

[0266] The present invention also relates to an extracellular vesicle that can be obtained by the process according to the invention.

[0267] Advantageously, the extracellular vesicle obtained by the process according to the invention comprises at least one marker selected from the group comprising CD9, CD63, CD81, ALIX and TSG101.

[0268] Advantageously, the inventors have demonstrated that the extracellular vesicle obtained by the process according to the invention comprises a significant increase in the concentration of selected proteins in the group comprising the target chromatin of prmtl (“chromatin target of

[0269] REPLACEMENT SHEET (RULE 26) prmtl (Chtop)), tissue factor pathway inhibitor (Tfpi), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (Adamts4), cysteine-rich angiogenic protein 61 (Ccn1) and neuron-derived neurotrophic factor (Ndnf)).

[0270] The inventors are the first to have demonstrated that extracellular vesicles obtained by the process according to the invention comprise biocompatible polymers on their surfaces, said polymers being attached to the extracellular vesicles, for example, by binding to cell adhesion proteins or membrane lipoproteins, with a very high affinity. The inventors have also demonstrated, surprisingly and unexpectedly, that the extracellular vesicles obtained, when advantageously used, directly target the tissue lesion and / or the lesion site. In other words, the extracellular vesicles combined with the biocompatible polymers according to the invention—for example, extracellular vesicles to which biocompatible polymers according to the invention are attached—when used to treat a tissue lesion, specifically target said lesion and / or are specifically addressed to said lesion.

[0271] The present invention also relates to an extracellular vesicle that can be obtained according to the process of the invention for use in the prevention and / or treatment of tissue injury.

[0272] The tissue injury is as defined above.

[0273] Brief description of the figures

[0274] Figure 1 shows photographs and curves of extracellular vesicles. Figure 1A shows transmission electron microscopy photographs after negative staining with 1.5% uranyl acetate. Figure 1B shows a concentration curve of

[0275] REPLACEMENT SHEET (RULE 26) Particles (particles / ml) (ordinate) present in the obtained medium as a function of their size (nm) (abscissa) according to the culture of mesenchymal stem cells (MSCs) without (control) or in the presence of a biocompatible polymer, namely OTR4132. Figure 1C represents a diagram corresponding to the size of extracellular vesicles (ordinate) obtained after culture of mesenchymal stem cells (MSCs) without (control) or in the presence of a biocompatible polymer, namely OTR4132, and the number of EVs. Figure 1D represents a diagram corresponding to the number of extracellular vesicles obtained (ordinate) after culture of mesenchymal stem cells (MSCs) without (control) or in the presence of a biocompatible polymer, namely OTR4132.Figure 1 E represents a diagram corresponding to the number of extracellular vesicles per cell (ordered) obtained after culture of mesenchymal stem cells (MSCs) without (control) or in the presence of biocompatible polymer, namely OTR4132.

[0276] Figure 2A represents a volcano plot of the expression of the 500 most variable genes; the ordinate corresponds to the negative base-10 logarithm of the calculated p-value, and the abscissa corresponds to the base-2 logarithm variation of the messenger RNA transcript expression observed between the two samples. Figure 2B represents a volcano plot of the protein concentration in extracellular vesicles obtained after culturing MSCs in the presence or absence of a biocompatible polymer, namely OTR4132; the ordinate corresponds to the negative base-10 logarithm of the calculated p-value, and the abscissa corresponds to the base-2 logarithm variation of the protein enrichment observed between the two samples.

[0277] Figure 3 shows the relative expression levels of the genes Chtop, Tfpi, Adamts4, Ccn1, and Ndnf in MSCs as a function of their culture in medium without or with OTR4132. Values ​​are presented as median ± IQR calculated from 5 independent experiments (Student's t-test). The y-axis represents the relative gene expression in MSCs normalized to housekeeping genes.

[0278] REPLACEMENT SHEET (RULE 26) Figure 4 shows a schematic representation of the proteins Tfpi (Figure 4A), Ccn1 (Figure 4B), Adamts4 (Figure 4C), and Ndnf (Figure 4D). The heparin-binding sites in Tfpi are one in the disordered C-terminal domain, which comprises a cluster of basic residues (HBS1), and the other in the 3rd BPTI / Kunitz inhibitor-like domain (HBS2). HBS stands for heparan-binding site, and the number corresponds to the site number.

[0279] Figure 5 shows the sensograms representing the surface plasmon resonance response (SPR) in RU as a function of time in seconds (s) for the 5 proteins: Human Basic Fibroblast Growth Factor 2 (hFGF2), tissue factor pathway inhibitor (hTFPI), cysteine-rich angiogenic protein 61 (hCCN1), chromatin target of prmtl (hCHTOP), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (hADAMTS44), human neuron-derived neurotrophic factor (hNDNF) and the positive control FGF-2.

[0280] Figure 6 illustrates the effect of a biocompatible polymer, OTR4132, on the localization of MSC cells in an ischemic area of ​​the brain. Figure 6A shows light microscopy images (Leica Microsystems DMi8) and a diagram after processing with ImageJ software (trademark) (Wayne Rasband, NI MH, Maryland, USA) (Schneider, Rasband, and Eliceiri, 2012). Figure 6A shows an image of MSC clustering in the ipsilateral hemisphere near the lesion site. Figure 6B shows an image of MSC clustering in the ipsilateral hemisphere near the lesion site after administration of OTR4132 and / or MSC 6B cells at different times. Figure 6C shows a diagram representing the surface area in mm². 2 BrdU-labeled in the ipsilateral hemisphere (ordered) measured by immunohistochemistry as a function of MSC administration in combination or not with the biocompatible polymer OTR4132 at different times after injury.

[0281] REPLACEMENT SHEET (RULE 26) Figure 7. Three-dimensional representations (Figures 7A and 7B) of the tertiary structure of human 7A collagen from two perspectives showing areas rich in basic amino acids, which have an overall positive charge, and areas rich in acidic amino acids, which have an overall negative charge at physiological pH. Positively charged areas are represented by black circles in the figure.

[0282] Other advantages may become apparent to the person skilled in the art upon reading the examples below, illustrated by the attached figures, given for illustrative purposes.

[0283] Examples

[0284] Example 1: Composition comprising biocompatible polymers and extracellular vesicles

[0285] A / Preparation of biocompatible polymers.

[0286] The synthesis of biocompatible polymers, RGTA, is described in the prior art, for example in US patent 7396923 entitled "Process for the sulfonation of compounds comprising free hydroxyl (OH) groups or primary or secondary amines"

[0015] and also in the bibliographic reference Yasunori I. et al., Biomaterials 2011, 32:769e776) and Petit E. et al., Biomacromolecules. 2004 Mar-Apr; 5(2):445-52

[0016] ,

[0287] Several RGTAs are known and described, and have been used, including OTR4120, which is described in numerous preclinical and clinical publications (RGTA®-based matrix therapy - A new branch of regenerative medicine in locomotion. Barritault D, Desgranges P, Meddahi-Pellé A, Denoix JM, Saffar JL. Joint Bone Spine. 2017 May;84(3):283-292. doi: 10.1016 / j.jbspin.2016.06.012 ([4], Barritault, et al., 10.1016 / j.jbspin.2016.06.012), RGTA® or ReGeneraTing Agents mimic heparan sulfate in regenerative medicine: from concept to curing patients. Barritault D, Gilbert-Sirieix M, Rice KL, Sineriz F, Papy-Garcia D, Baudouin C, Desgranges P, Zakine G, Saffar JL, van Neck J. Glycoconj J. 2017 Jun;34(3):325-338. doi: 10.1007 / sl 0719-016-9744-5 ([3], Barritault, et al., 10.1007 / sl 0719-016-9744-5).

[0288] REPLACEMENT SHEET (RULE 26) Compound OTR4131 is a compound comprising a Z radical which is a fatty acid, namely acetic acid as described in Frescaline G. et al., Tissue Eng Part A. 2013 Jul; 19(13-14): 1641-53. doi:

[0289] 10.1089 / ten.TEA.2012.0377 (

[0042] , Frescaline, et al.,

[0290] 10.1089 / ten.TEA.2012.0377), Randomized controlled trial demonstrates the benefit of RGTA® based matrix therapy to treat tendinopathies in racing horses. Jacquet-Guibon S, Dupays AG, Coudry V, Crevier-Denoix N, Leroy S, Sineriz F, Chiappini F, Barritault D, Denoix JM. PLoS One. 2018 Mar 9;13(3):e0191796. doi: 10.1371 / journal.pone.0191796 (

[0043] , Jacquet-Guibon, et al., 10.1371 / journal.pone.0191796). Other compounds are also described in patent documents US06689741 (

[0044] , US06689741),

[0291] US2014301972A1 (

[0045] , US2014301972A1) in which Z is an amino acid such as phenylalanine (Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Holmes BB, DeVos SL, Kfoury N, Li M, Jacks R, Yanamandra K, Ouidja MO, Brodsky FM, Marasa J, Bagchi DP, Kotzbauer PT, Miller TM, Papy-Garcia D, Diamond Ml. Proc Natl Acad Sci US A. 2013 Aug 13;110(33):E3138-47. doi: 10.1073 / pnas.1301440110 (

[0046] , Holmes, et al., 10.1073 / pnas.1301440110)) or other hydrophobic compound (Structure-activity studies of heparan mimetic polyanions for anti-prion therapies. Ouidja MO, Petit E, Kerros ME, Ikeda Y, Morin C, Carpentier G, Barritault D, Brugère-Picoux J, Deslys JP, Adjou K, Papy-Garcia D. Biochem Biophys Res Commun. 2007 Nov 9;363(1):95-100 (

[0047] , Ouidja, et al., 10.1016 / j.bbrc.2007.08.113)).

[0292] B / Preparation of extracellular vesicles

[0293] In this example, the cells used were mesenchymal stem cells (MSCs) extracted from the femurs and tibias of Sprague-Dawley rats, male rats aged 8 to 10 weeks, Janvier Labs, France. The animals were anesthetized by inhalation of 5% isoflurane in an O2 / N2O mixture (1 / 3 ratio) and then killed by CO2 inhalation. After dissection of the femurs and tibias under a laminar flow hood, they were placed in an alcohol bath, followed by a penicillin-streptomycin bath, and then rinsed.

[0294] REPLACEMENT SHEET (RULE 26) in 1X saline buffer. The epiphyses were sectioned and the bone marrow was collected in Dulbecco's Modified Eagle's Medium (DMEM) - low glucose (commercial reference D4655, marketed by Sigma-Aldrich) by back-and-forth insertion into the bone lumen using a 21-gauge needle attached to a 5 mL syringe containing 3 mL of culture medium. The collected contents were then centrifuged for 5 min at 300 x g.The cell pellets were resuspended in 10 mL of Dulbecco's Modified Eagle's Medium (DMEM) - low glucose (commercial reference D4655, marketed by Sigma-Aldrich), supplemented with 1% penicillin-streptomycin (commercial reference P4333, marketed by Sigma-Aldrich), 2 mM L-glutamine (commercial reference G7513, marketed by Sigma-Aldrich), and 20% fetal bovine serum (commercial reference FB-1001, marketed by Biosera). The cells were then cultured in 75 cm³ culture dishes. 2 (T75). The cells were incubated under a humid atmosphere composed of 95% air and 5% CO2 at 37°C. After 3 to 4 days, the 75 cm³ culture dishes 2(T75) were gently shaken, rinsed twice with Phosphate Buffer Saline (PBS) solution, and then the medium was replaced with complete medium similar to that used previously. This eliminated non-adherent hematopoietic cells, dead cells, and cellular debris, unlike the MSCs, which adhered to the bottom of the T75 medium. The medium was changed every 3 to 4 days, and the mesenchymal stem cells (MSCs) were detached from the cell mat by trypsinization when they reached 90% confluence, washed, and diluted for reseeding at a cell density of 1 x 10⁻⁶. 6 cells in a new 75 cm flask 2T75 in Dulbecco's Modified Eagle's Medium (DMEM) - low glucose culture medium (commercial reference D4655, marketed by Sigma-Aldrich), supplemented with 1% penicillin-streptomycin (commercial reference P4333, marketed by Sigma-Aldrich), 2 mM L-glutamine (commercial reference G7513,

[0295] REPLACEMENT SHEET (RULE 26) marketed by Sigma-Aldrich) and 10% fetal calf serum (commercial reference FB-1001, marketed by Biosera).

[0296] MSCs were maintained in culture at 3 èmeThe culture medium was transferred to T75 flasks until it reached 80% confluence, after which it was washed. 0.1 pg / mL of biocompatible polymer, namely OTR4132, was added to the culture medium, which was then decanted into fetal bovine serum (FBS). After 24 h of culture in the OTR4132-containing medium, 240 mL of the conditioned medium (with or without 0.1 pg / mL of OTR4132) from the culture of 24 MSC T75 flasks was collected and centrifuged twice at 4 °C: 2,000 x g for 20 min and 10,000 x g for 30 min, to remove dead cells and cellular debris. The supernatant was then filtered through a 0.22 µm membrane (Sarstedt) and centrifuged at 100,000 x g (34,000 rpm) (Beckman Coulter) for 70 min at 4 °C according to the procedure described in Théry et al., 2006, and Xu et al., 2019, (

[0041] , Théry, et al., 10.1002 / 0471143030.cb0322s30) (

[0040] , Xu, et al., 10.1080 / 08958378.2019.1597220).The pellet was washed with Phosphate Buffer Saline (commercial reference CS1 PBS01 K BP, marketed by Eurobio) PBS and subjected to a second ultracentrifugation at 100,000 x g (34,000 rpm, Beckman Coulter) for 70 min at 4°C. The resulting pellet contained extracellular vesicles with or without biocompatible polymer, which were resuspended in Phosphate Buffer Saline (commercial reference CS1 PBS01 K BP, marketed by Eurobio) PBS 1X and stored at -80°C for subsequent analyses. The solutions thus obtained contained the extracellular vesicles.

[0297] A study of the extracellular vesicles (EVs) thus obtained was performed. An evaluation of EVs obtained from MSCs cultured in the presence of OTR4132 showed differences compared to those from MSCs cultured without OTR4132. The following parameters were studied on the isolated EVs:

[0298] - their quantity and size, namely the average diameter, using a vesicle analyzer (NanoSight NS300) and electron microscopy;

[0299] REPLACEMENT SHEET (RULE 26) - their genetic material content using the RNAseq technique;

[0300] - their protein content and their interaction with OTR4132 using;

[0301] - proteomics;

[0302] - the in silico identification of TOTR4132 binding sites;

[0303] - surface plasmon resonance (SPR).

[0304] Figure 1 shows the different results obtained. Figure 1A shows transmission electron microscopy (TEM) photographs. 4 µL of pure EVs were deposited on a carbon / formvar / copper grid, incubated for 5 min at room temperature (20°C), and capillary-dried on Whatman paper. To fix and stain the EVs, a drop of 1.5% uranyl acetate (supplier reference 73943-25G Honeywell Fluka 25GR, Thermo Fisher Scientific) was placed on the grid, incubated for 15 sec at room temperature (20°C), and again capillary-dried on Whatman paper. The EVs were then visualized using a JEOL JEM 1011 TEM 100kV transmission electron microscope (JEOL USA, Inc., Peabody, MA) with an Orius SC200 CCD camera (Gatan, USA). As shown in these photographs, the extracellular vesicles obtained have a similar diameter: 147.33 nm and 177.30 nm, respectively.Sample concentrations and EV sizes were measured using a NanoSight NS300 (Malvern Panalytical) with a 405 nm laser. Samples were diluted in 1X PBS (Supplier reference 11503387 Gibco™ PBS, pH 7.4, FisherScientific) to obtain acceptable concentrations according to the manufacturer's recommended use of the instrument, namely 3.10. 8 at 5.10 8 VE / mL. The instrument was rinsed with 1X PBS between each analysis. NTA 3.3 software (Malvern Panalytical) was used to process and analyze the five 60-second videos recorded per sample analyzed with the NanoSight NS300. Figure 1B shows plots of particle concentration (particles / ml) in the obtained medium as a function of particle size (nm) in mesenchymal stem cell (MSC) culture with or without a biocompatible polymer, namely

[0305] REPLACEMENT SHEET (RULE 26) OTR4132. As demonstrated, the samples included EVs with a peak size around 100–150 nm, characteristic of exosomes. Figure 1C shows a diagram corresponding to the size of extracellular vesicles obtained after culturing mesenchymal stem cells (MSCs) without (control) or in the presence of the biocompatible polymer, namely OTR4132. As demonstrated, OTR4132 in the culture medium does not alter the size of EVs obtained from MSCs, with a median size in both conditions around 130 nm. Figure 1D shows a diagram corresponding to the number of extracellular vesicles obtained after culturing mesenchymal stem cells (MSCs) without (control) or in the presence of the biocompatible polymer, namely OTR4132. As demonstrated, TOTR4132 increases the number of EVs present in the samples.Figure 1E represents a diagram corresponding to the number of extracellular vesicles per cell obtained after culture of mesenchymal stem cells (MSCs) without (control) or in the presence of biocompatible polymer, namely TOTR4132. As demonstrated, the presence of TOTR4132 in the culture medium significantly increases the number of EVs secreted / obtained from MSCs.

[0306] The results clearly demonstrate that no difference in the number of MSCs was observed depending on the culture conditions (in the presence or absence of OTR4132). The results also clearly demonstrate that the number of extracellular vesicles obtained was twice as high in the presence of OTR4132 without any change in the size and / or morphology of said extracellular vesicles (Figure 1 A and B).

[0307] Figure 1E below illustrates this significant difference in the number of EVs / cells. (Student's t-test, p = 0.0286).

[0308] A proteomic analysis of the extracellular vesicles obtained was also performed by mass spectrometry. In order to digest the samples, they were incubated in the presence of 20% SDS (Supplier reference 436143, Sigma-Aldrich, Merck) to a final concentration of 5%, reduced with 20 mM TCEP (tris(2-carboxyethyl)phosphine hydrochloride) (Supplier reference: C4706, Sigma-Aldrich,

[0309] REPLACEMENT SHEET (RULE 26) Sigma-Aldrich, Merck,) and alkylated with 50 mM CAA (chloroacetamide) (Supplier part number: 22790, Sigma-Aldrich, Merck) for 5 min at 95 °C. Aqueous phosphoric acid (Supplier part number 1.00573, Sigma-Aldrich, Merck) was added to a final concentration of 2.5%, followed by the addition of S-Trap bonding buffer (90% aqueous methanol, 100 mM TEAB, pH 7.1) Supplier part numbers 34860 ​​and 241059, Sigma-Aldrich, Merck. The mixtures were then loaded onto S-Trap™ columns (marketed by ProtiFi, USA). Five washes were performed for complete removal of SDS. The samples were digested with 1.5 pg of trypsin (Promega) at 47 °C for 1 h.After elution, the peptides were vacuum-dried and resuspended in 2% acetonitrile (ACN) (Supplier reference 439134, Sigma-Aldrich, Merck) and 0.1% formic acid (Supplier reference W248703, Sigma-Aldrich, Merck) in water for high-performance liquid chromatography (HPLC) analysis prior to mass spectrometry. The tryptic peptides were resuspended in 30 pL, and a volume corresponding to 400 ng for each sample was injected into an HPLC system (Bruker Daltonics, Germany) coupled to a timsTOF Pro mass spectrometer (Bruker Daltonics, Germany).HPLC separation (solvent A: 0.1% formic acid in water, 2% acetonitrile; solvent B: 0.1% formic acid in ACN) was carried out at 250 nL / min using an emitter column (C18, 25 cm x 75 pm x 1.6 pm) (Ion Optics, Australia) using a 40 min elution gradient (2 to 11% solvent B for 19 min; 11 to 16% for 7 min; 16% to 25% for 4 min; 25% to 80% for 3 min and finally 80% for 7 min to wash the column). Mass spectrometry data were acquired using the parallel accumulation serial fragmentation acquisition (PASEF (registered trademark)) method with data-dependent acquisition (DDA) mode. Measurements were performed with a mass-to-charge ratio of 100 to 1700 thomson (Th). The obtained data were analyzed using version 2.0.1.0 of the MaxQuant software and searched using the Andromeda search engine.

[0310] REPLACEMENT SHEET (RULE 26) UniProtKB / Swiss-Prot Rattus norvegicus database mixed with its TrEMBL entries (updated February 2021, 48085 entries). Scores were calculated in MaxQuant as previously described (

[0048] , Cox, et al., 10.1038 / nbt.1511). Statistical and bioinformatic analyses, including heat maps, profile plots, and clustering, were performed using Perseus software (version 1.6.14.0, MaxQuant). For statistical comparison, two groups were defined: treated and untreated, each containing three biological replicates, with or without OTR4132. A Student's t-test was performed, and the type I error (α) was set at 0.05. The results of this analysis demonstrated that the extracellular vesicles obtained, regardless of the culture conditions of the MSCs, expressed the following markers CD9, CD63, CD81, ALIX and TSG101 which are specific to exosomes.Furthermore, as demonstrated in Figure 2A, which represents a volcano plot concerning the expression of the 500 most variable genes, no difference in expression was identified depending on the culture in the presence or absence of a biocompatible polymer, namely OTR4132.

[0311] However, as demonstrated in Figure 2B and Table 1 below, representing respectively a Volcano presentation diagram and a table of protein concentration in extracellular vesicles obtained after culturing MSCs in the presence or absence of a biocompatible polymer, namely OTR4132.

[0312] Table 1: Variation factor in log base 2.

[0313] REPLACEMENT SHEET (RULE 26)

[0314] As demonstrated, in the presence of OTR4132 a significant increase in the concentration in extracellular vesicles of prmtl target chromatin (“chromatin target of prmtl” (Chtop)), tissue factor pathway inhibitor” (Tfpi)), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (“disintegrin and metalloproteinase with thrombospondin type 1 motif 4” Adamts4), cysteine-rich angiogenic protein 61” (Ccn1) and neuron-derived neurotrophic factor” (Ndnf)) was observed. In particular, the increase in concentration in extracellular vesicles of Chtop, Tfpi, Adamts4, Ccn1 and Ndnf was respectively 6, 7, 15, 50 and 80 times in extracellular vesicles, to have exosomes, as shown in Figure 2B.

[0315] An analysis of the expression of the Chtop, Tfpi, Adamts4, Ccn1, and Ndnf genes in the MSCs was also performed. The mRNA from the MSCs from which the EVs were isolated was extracted and purified using the RNeasy Micro kit (Supplier reference 74104, Qiagen, Courtaboeuf, France). The mRNA concentration was measured with the NanoDrop™ 2000 spectrophotometer (trademark) (Supplier reference ND-2000, Thermo Fisher Scientific, USA). The mRNAs were then reverse-transcribed with the iScript reverse transcription supermix (Bio-Rad), and the cDNA was amplified with the CFX96 Touch real-time PCR detection system (Bio-Rad) and the iTaq Universal SYBR® Green supermix (Bio-Rad) using primers designed by Eurogentec (see Table 2 below). Glyceraldehyde-3-phosphate dehydrogenase (Gapdh), cyclophilin A and actin (Act) were used as controls for endogenous RNA (household genes).Primer sequences for the genes neuron-derived neurotrophic factor (Ndnf), chromatin target of prmtl (Chtop), tissue factor pathway inhibitor (Tfpi), disintegrin and metalloproteinase with.

[0316] REPLACEMENT SHEET (RULE 26) thrombospondin type 1 motif 4 (Adamts4) and cysteine-rich angiogenic protein 61 Ccn1) were purchased from Eurogentec as described in Table 2 below.

[0317] Table 2: Primer sequences allowing analysis of the expression of housekeeping genes and genes of interest encoding proteins identified in extracellular vesicles of hematopoietic mesenchymal stromal cells treated with OTR4132.

[0318] The genes listed in Table 2 above refer to rat genes. Samples were analyzed in triplicate using the following amplification protocol: 95°C for 3 min followed by 40 cycles at 95°C for 3 sec and 60°C for 30 sec. Expression levels were calculated using the comparative Ct (ACt) method after normalization with

[0319] REPLACEMENT SHEET (RULE 26) The housekeeping genes Gapdh, Actin, and cyclophilin A. The results are expressed as relative gene expression normalized to the mean of the housekeeping genes (cyclophilin A, actin, and GAPDH). Figure 3 shows the relative gene expression levels in MSCs as a function of their culture in medium without or with OTR4132. The values ​​are shown as median ± IQR calculated from 5 independent experiments (Student's t-test). As shown in Figure 3, the results clearly demonstrate no difference in expression based on the MSC culture conditions.

[0320] These results clearly demonstrate that the combination of extracellular vesicles and an example of a biocompatible polymer advantageously protects the chromatin target of prmtl (Chtop), tissue factor pathway inhibitor (Tfpi), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (Adamts4), cysteine-rich angiogenic protein 61 (Ccn1) and neuron-derived neurotrophic factor (Ndnf).

[0321] The proteins tfpi, ccn1, and adamts4 are known to have heparin-binding sites, as shown in Figure 4. Two heparin-binding sites have been described in tfpi: one in the disordered C-terminal domain, which includes a cluster of basic residues (HBS1), and the second in the third BPTI / Kunitz inhibitor-like domain (HBS2), identified by comparing the NMR structures of this domain with and without heparin (

[0049] , Mine, et al., 10.1021 / bi011299g). A heparin-binding site has been described in ccn1 in its cysteine-knotted domain, which also has a cluster of basic residues (

[0050] , Chen, et al., 10.1074 / jbc.M003040200). Four heparin-binding sites have been described in adamts4. The site described in the type I thrombospondin domain was identified by homology with the heparin-binding site found in adamts1 (HBS1). Three heparin-binding sites have also been identified in the cysteine-enriched domain.

[0322] REPLACEMENT SHEET (RULE 26) (HBS2) and in the spacer domain (HBS3 and HBS4), peptides corresponding to their sequence inhibit the heparin binding of adamts4 (

[0051] , Jones, et al., 10.1186 / ar1783). No heparin-binding site has been described in Ndnf and Chtop.

[0323] An affinity analysis was performed for the proteins chromatin target of prmtl (Chtop), tissue factor pathway inhibitor (Tfpi), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (Adamts4), cysteine-rich angiogenic protein 61 (Ccn1), and neuron-derived neurotrophic factor (Ndnf) with a biocompatible polymer, such as OTR4132. Affinity determination was carried out using Surface Plasmon Resonance (SPR) techniques on a Biacore™ T200 instrument (Cytiva) at 25°C. Biotin was grafted onto the OTR4132 molecule using the free aldehyde at the end of the glycosidic chain, according to the procedure described below. The method chosen to graft biotin onto RGTA® is a nitrogen-initiated addition-elimination of biotin acylhydrazide to the reducing end of the OTR4132 chain.A bound acylhydrazone (R-CO-NH-N=R') was created by the non-participating electron pair (CH=N:) and an acidic amino nitrogen atom (-NH-)[10.3390 / molecules27248719]. An excess of biotin acylhydrazide was used relative to the free terminal aldehydes of OTR4132. Biotinylated OTR4132 was captured on several surfaces of an SA S-series detection chip via its interaction with streptavidin previously fixed to the Biacore™ chip (SA, S-series, Cytiva). To measure interaction kinetics and affinities, analyte samples corresponding to the 5 proteins (hTFPI, hADAMTS4, hCCN1, hCHTOP, hNDNF) were injected in duplicate at 5 serial concentrations every 300 seconds.The proteins were injected at 5 increasing concentrations, namely 0.312 nM, 0.625 nM, 1.25 nM, 2.5 nM, and 5 nM for hFGF2; 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, and 10 nM for hTFPI, hCCN1, and hCHTOP; and 6.25 nM, 12.5 nM, 25 nM, 50 nM, and 100 nM for hADAMTS4 and hNDNF, every 300 s. These injections were performed using the Single Cycle Kinetics strategy in a constant flow of 30 pL / mL saline (Hepes buffered saline, HBS-EP+, Cytiva).

[0324] REPLACEMENT SHEET (RULE 26) The protein hFGF2, known to interact with TOTR4132 (internal data), was used as a positive control because it possesses a heparan sulfate binding site. The dissociation phase was measured by injecting saline solution (HBS-EP+, 10X (Cytiva ref: BR100669) Lot# BCBW6092) for 1200 seconds. Between duplicates, analytes bound to TOTR4132 were removed with 2M NaCl (for hTFPI, hCCN1, hCHTOP, and hNDNF) or 0.25% sodium dodecyl sulfate (SDS) (for hADAMTS4). Data analysis was performed using Biacore™ Insight Evaluation software (Cytiva).For each protein, the mean and standard error (SD) values ​​of the association constant (ka), dissociation constant (kd), and affinity constant (KD = kd / ka) were measured. If the ka and kd values ​​could not be determined, then the KD was calculated from the saturation curve, which corresponds to the concentration value on the x-axis equal to RUmax / 2. The results are shown in Figure 5, which includes (A) sensograms representing the SPR response over time for the five proteins and the FGF-2 positive control. The proteins were injected at five increasing concentrations: 0.312 nM, 0.625 nM, 1.25 nM, 2.5 nM, and 5 nM for hFGF2; 0.625 nM, 1.25 nM, 2.5 nM, and 10 nM for hTFPI; hCCN1 and hCHTOP and 6.25 nM; 12.5 nM; 25 nM; 50 nM and 100 nM for hADAMTS4 and hNDNF every 300 s.The interaction was assessed by measuring ka, kd, and the calculated KD (KD = kd / ka). The results are shown in Table 3 below. The data represent the mean ± standard deviation (SD) of the replicates.

[0325] Table 3: Results for the values ​​of ka, kd and KD

[0326] REPLACEMENT SHEET (RULE 26)

[0327] In the table, nd means not determined.

[0328] Figure 5 shows the measured affinity of these proteins to an example of a biocompatible polymer, for example OTR4132. As a comparator, the affinity of recombinant human FGF2 for RGTA is given as an affinity constant (KD) of 8.24 nM (8.24 x 10⁻¹⁰). 9 As shown in Figure 5, the strongest affinity was determined for the hCcnl protein (KD not measurable), and the affinities measured for the other 4 proteins are very strong, namely 3.73 x 10 12for hCHTOP, 2.52 x 10 8 for hTFPI, 1.52 x10 9 for hADAMTS4 and 5.40 x 10' 10 for hNDNF clearly demonstrating a bond between said proteins and the biocompatible polymer, for example OTR4132.

[0329] These results clearly demonstrate that the biocompatible polymer, for example TOTR4132, through strong binding to proteins on extracellular vesicles, exhibits a universal mechanism of tissue repair and regeneration. By this mechanism, the biocompatible polymer, such as TOTR4132, facilitates the delivery of extracellular vesicles to areas with accessible binding sites for biocompatible polymers, such as TOTR4132. In particular, it is known that biocompatible polymers, such as TOTR4132, bind to sites where endogenous heparan sulfates have been degraded during tissue damage, notably by glycanases, and replace these degraded endogenous heparan sulfates, which are not degraded by glycanases. Thus, this example

[0330] REPLACEMENT SHEET (RULE 26) clearly demonstrates that the combination of extracellular vesicles and biocompatible polymer according to the invention, in particular by means of a strong attachment of an example of biocompatible polymer, namely OTR4132, to proteins on the extracellular vesicles makes it possible to target said extracellular vesicles to lesion sites.

[0331] This example clearly demonstrates that the combination of a biocompatible polymer and extracellular vesicles advantageously allows for the delivery of these extracellular vesicles to the site of injury, regardless of their cellular origin. In particular, this example clearly demonstrates that the biocompatible polymer binds to the extracellular vesicles and advantageously protects them and / or the proteins to which the biocompatible polymer is bound from glycanases. This example also clearly demonstrates that the combination of a biocompatible polymer and extracellular vesicles advantageously allows for the targeting of tissue lesions, for example, by binding to heparan sulfate binding sites.

[0332] Example 2: Treatment of tissue damage with a pharmaceutical composition comprising a biocompatible polymer and extracellular vesicles

[0333] In this example, the lesion was a rat brain lesion due to ischemia according to the procedure described in Khelif et al., 2018, DOI: 10.7150 / thno.28252

[0028] ,

[0334] In this example, the biocompatible polymer was OTR4132, marketed by OTR3 Paris France. Mesenchymal stem cells (MSCs) from the bone marrow of Sprague Dawley rat femurs and tibias, weighing 300 to 350 grams, were used. The MSCs were pre-labeled with bromodeoxyuridine (BrdU, 3 mg / mL, Sigma) by daily addition to the MSC culture medium for 3 days (

[0052] , Jin, et al., 10.1155 / 2016 / 8616143) (

[0053] , Jiang, et al., 10.1007 / s13770-021-00421-5). The resulting labeled cells were injected intravenously (IV) 3 x 10⁻¹³ 6 cells in 300 pl

[0335] REPLACEMENT SHEET (RULE 26) of PBS1X (Supplier Reference 11503387, Gibco™ PBS, pH 7.4, Fisher Scientific) alone or in mixture with the biocompatible polymer OTR4132. The concentration of said biocompatible polymer OTR4132 in the injected composition was 1.5 mg / kg diluted in 0.9% sterile saline water for injection (NaCl 0.9% sterile solution for injection, COOPER; Ref: 340093509484; Physiological Serum, OMNIA, Ref: 32.E0001).

[0336] A histochemical analysis to identify and quantify BrdU in the whole brain was performed two days after induction of cerebral ischemia. The sections were washed in PBS1X, incubated in 2N HCl at 37°C for 1 h, followed by a 10-minute incubation in 0.1 M boric acid. The sections were then blocked in 3% BSA (bovine serum albumin, Thermo Scientific™ Blocker™ BSA 10% Ref: 1799236, diluted one-third in PBS1X) for 2 h at room temperature, i.e. 20 °C, and stained with a rat polyclonal anti-BrdU antibody (Roche 0.2 pg / mL, Roche 0.2 pg / mL, Cell Proliferation ELISA, BrdU (colorimetric), Ref: 11 647 229 001) overnight at 4 °C.The sections were then washed with PBS1X (at least 3 times, Gibco™ PBS, pH 7.4, FisherScientific Ref: 11503387), followed by a 2 h incubation with the secondary antibody Alexa Fluor® 488 (4 pg / mL, Invitrogen) and the nuclear marker Hoechst 33342 (1 / 5000, Sigma Aldrich) at room temperature, i.e. 22 °C.

[0337] Images were obtained using an optical microscope (Leica microsystems DMi8). All image processing and analysis were performed using Imaged® software (Wayne Rasband, NIMH, Maryland, USA) (Schneider, Rasband, and Eliceiri, 2012). Figure 6 shows the results. As demonstrated in Figure 6, clusters of MSCs were predominantly found in the ipsilateral hemisphere near the lesion site (Figures 6A and 6B). Combining the biocompatible polymer OTR4132 with MSCs significantly increased the number of MSCs localized in the ipsilateral hemisphere near the lesion site compared to the administration of cells alone (Figures 6B and 6C; Student's t-test; p-value = 0.0059).

[0338] REPLACEMENT SHEET (RULE 26) Reference List

[0339] [1]. US8790631. Use of biocompatible polymers for the preparation of a composition or a medical device.

[0340] [2], W02020151900. Cosmetic / dermatological composition

[0341] [3], Barritault, D., Gilbert-Sirieix, M., Rice, K. L., Sineriz, F., Papy-Garcia, D.,

[0342] Baudouin, C., Desgranges, P., Zakine, G., Saffar, J. L., and van Neck, J. RGTA((R)) or ReGeneraTing Agents mimic heparan sulfate in regenerative medicine: from concept to curing patients. Glycoconj J. 2017. 34. 10.1007 / sl 0719-016-9744-5

[0343] [4], Barritault, D., Desgranges, P., Meddahi-Pelle, A., Denoix, J. M., and

[0344] Saffar, J. L. RGTA((R))-based matrix therapy - A new branch of regenerative medicine in locomotion. Joint Bone Spine. 2017. 84. 10.1016 / j.jbspin.2016.06.012

[0345] [5], EP3302523B1. Composition for the treatment of tissue lesions 2016.

[0346] [6], Meddahi, A., Bree, F., Papy-Garcia, D., Gautron, J., Barritault, D., and

[0347] Caruelle, J. P. Pharmacological studies of RGTA(11), a heparan sulfate mimetic polymer, efficient on muscle regeneration. J Biomed Mater Res. 2002. 62. 10.1002 / jbm.10283

[0348] [7], Pereira, P. M., Papy-Garcia, D., Barritault, D., Chiappini, F., Jackisch, R.,

[0349] Schimchowitsch, S., and Cassel, J. C. Protective Effects of a synthetic glycosaminoglycan mimetic (OTR4132) in a rat immunotoxic lesion model of septohippocampal cholinergic degeneration. Glycoconj J. 2022. 39. 10.1007 / sl 0719-022-10047-x

[0350] [8], Xin, H., Li, Y., Cui, Y., Yang, J. J., Zhang, Z. G., and Chopp, M. Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats. J Cereb Blood Flow Metab. 2013. 33. 10.1038 / jcbfm.2013.152

[0351] [9], Doeppner, T. R., Herz, J., Gorgens, A., Schlechter, J., Ludwig, A. K.,

[0352] Radtke, S., de Miroschedji, K., Horn, P. A., Giebel, B., and Hermann, D. M. Extracellular Vesicles Improve Post-Stroke Neuroregeneration and

[0353] FEUILLE DE REMPLACEMENT (RÈGLE 26) Prevent Postischemic Immunosuppression. Stem Cells Transi Med.

[0354] 2015. 4. 10.5966 / sctm.2015-0078

[0355]

[0010] , Jeyaram, A., and Jay, S. M. Preservation and Storage Stability of

[0356] Extracellular Vesicles for Therapeutic Applications. AAPS J. 2017. 20. 10.1208 / S12248-017-0160-y

[0357]

[0011] , Ma, Z. J., Yang, J. J., Lu, Y. B., Liu, Z. Y., and Wang, X. X. Mesenchymal stem cell-derived exosomes: Toward cell-free therapeutic strategies in regenerative medicine. World J Stem Cells. 2020. 12.

[0358] 10.4252 / wjsc.v12.i8.814

[0359]

[0012] , Yanez-Mo, M., Siljander, P. R., Andreu, Z., Zavec, A. B., Borras, F. E.,

[0360] Buzas, EI, Buzas, K., Casai, E., Cappello, F., Carvalho, J., Colas, E., Cordeiro-da Silva, A., Fais, S., Falcon-Perez, JM, Ghobrial, IM, Giebel, B., Gimona, M., Graner, M., Gursel, I., Gurselga, N.H. Hendrix, A., Kierulf, P., Kokubun, K., Kosanovic, M., Kralj-lglic, V., Kramer-Albers, E.M., Laitinen, S., Lasser, J., Lehner, T., Ligeti, E., Line, A., Lipps, G., Llorente, A., Lotvall, M. Keber, Mancek, M. Keber. A., Mittelbrunn, M., Nazarenko, I., Nolte-'t Hoen, EN, Nyman, TA, O'Driscoll, L, Olivan, M., Oliveira, C., Pallinger, E., Del Portillo, HA, Reventes, J., Rigau, M., Rohde, E., Sammar, M., Sanchez, F., Madrid, Madrid. N., Schallmoser, K., Ostenfeld, MS, Stoorvogel, W., Stukelj, R., Van der Grein, SG, Vasconcelos, MH, Wauben, MH, and De Wever, O. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015. 4. 10.3402 / jev.v4.27066

[0361]

[0013] , Hallal, S., Tuzesi, A., Grau, G. E., Buckland, M. E., and Alexander, K. L.

[0362] Understanding the extracellular vesicle surface for clinical molecular biology. J Extracell Vesicles. 2022. 11. 10.1002 / jev2.12260

[0363]

[0014] , Buzas, E. I., Toth, E. A., Sodar, B. W., and Szabo-Taylor, K. E. Molecular interactions at the surface of extracellular vesicles. Semin Immunopathol. 2018. 40. 10.1007 / S00281 -018-0682-0

[0364]

[0015] , Kalimuthu, K., Kwon, W. Y., and Park, K. S. A simple approach for rapid and cost-effective quantification of extracellular vesicles using a

[0365] FEUILLE DE REMPLACEMENT (RÈGLE 26) fluorescence polarization technique. J Biol Eng. 2019. 13.

[0366] 10.1186 / S13036-019-0160-9

[0367]

[0016] , Uddin, M. J., Mohite, P., Munde, S., Ade, N., Oladosu, T. A., Chidrawar,

[0368] V. R., Patel, R., Bhattacharya, S., Paliwal, H., and Singh, S. Extracellular vesicles: The future of therapeutics and drug delivery systems. Intelligent Pharmacy. 2024. 2. 10.1016 / j.ipha.2024.02.004

[0369]

[0017] , Fields, G. B. Introduction to peptide synthesis. Curr Protoc Protein Sci.

[0370] 2002. Chapter 18. 10.1002 / 0471140864.ps1801s26

[0371]

[0018] , Chan, W. C., and White, P. D. (2000) Fmoc solid phase peptide synthesis

[0372] : a practical approach, Oxford University Press, New York A 0199637253 (hb)

[0373] 0199637245 (pbk.)

[0374]

[0019] , Benoiton, L. N. (2005) Chemistry of Peptide Synthesis 9780429119583

[0375]

[0020] , Sanlaville, C., Charmot, D., Petsko, G. A., and Ringe, D. (2008) Structure et fonction des protéines, De Boek University9782804158880

[0376]

[0021] , Raposo, G., and Stoorvogel, W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013. 200.

[0377] 10.1083 / jcb.201211138

[0378]

[0022] , Boireau, W., and Elie-Caille, C. Les vésicules extracellulaires. Med Sci

[0379] (Paris). 2021. 37. 10.1051 / medsci / 2021201

[0380]

[0023] , Battistelli, M., and Falcieri, E. Apoptotic Bodies: Particular Extracellular

[0381] Vesicles Involved in Intercellular Communication. Biology (Basel). 2020. 9. 10.3390 / biology9010021

[0382]

[0024] , Tan, Z. L, Li, J. F., Luo, H. M., Liu, Y. Y., and Jin, Y. Plant extracellular vesicles: A novel bioactive nanoparticle for tumor therapy. Front Pharmacol. 2022. 13. 10.3389 / fphar.2022.1006299

[0383]

[0025] , Takahashi, K., and Yamanaka, S. A decade of transcription factor- mediated reprogramming to pluripotency. Nat Rev Mol Cell Biol. 2016. 17. 10.1038 / nrm.2016.8

[0384]

[0026] , Maury, Y., Gauthier, M., Peschanski, M., and Martinat, C. [Human pluripotent stem cells: opening key for pathological modeling], Med Sci (Paris). 2011. 27. 10.1051 / medsci / 2011274023

[0385] FEUILLE DE REMPLACEMENT (RÈGLE 26)

[0027] , Caldwell, E. E., Nadkarni, V. D., Fromm, J. R., Linhardt, R. J., and Weiler,

[0386] J. M. Importance of specific amino acids in protein binding sites for heparin and heparan sulfate. Int J Biochem Cell Biol. 1996. 28. 10.1016 / 1357-2725(95)00123-9

[0387]

[0028] , Hileman, R. E., Fromm, J. R., Weiler, J. M., and Linhardt, R. J.

[0388] Glycosaminoglycan-protein interactions: definition of consensus sites in glycosaminoglycan binding proteins. BioEssays. 1998. 20.

[0389] 10.1002 / (SICI)1521 -1878(199802)20:2<156::AID-BIES8>3.0.CO;2-R

[0390]

[0029] , Wu, H.-f., Lundblad, R. L., and Church, F. C. Neutralization of Heparin

[0391] Activity by Neutrophil Lactoferrin. Blood. 1995. 85.

[0392] 10.1182 / blood.V85.2.421.421

[0393]

[0030] , Andersson, E., Rydengard, V., Sonesson, A., Morgelin, M., Bjorck, L., and Schmidtchen, A. Antimicrobial activities of heparin-binding peptides. Eur J Biochem. 2004. 271. 10.1 111 / j.1432- 1033.2004.04035.x

[0394]

[0031] , Sobel, M., Soler, D. F., Kermode, J. C., and Harris, R. B. Localization and characterization of a heparin binding domain peptide of human von Willebrand factor. Journal of Biological Chemistry. 1992. 267. 10.1016 / S0021 -9258(19)50359-3

[0395]

[0032] , Capila, I., and Linhardt, R. J. Heparin-Protein Interactions. Angewandte

[0396] Chemie International Edition. 2002. 41. 10.1002 / 1521 -

[0397] 3773(20020201 )41 :3<390::AID-ANIE390>3.0.CO;2-B

[0398]

[0033] , Munoz, E. M., and Linhardt, R. J. Heparin-binding domains in vascular biology. Arterioscler Thromb Vase Biol. 2004. 24.

[0399] 10.1161 / 01 .ATV.0000137189.22999.3f

[0400]

[0034] , UniProt, C. UniProt: the Universal Protein Knowledgebase in 2023.

[0401] Nucleic Acids Res. 2023. 51. 10.1093 / nar / gkac1052

[0402]

[0035] , Akbar, A., Malekian, F., Baghban, N., Kodam, S. P., and Ullah, M.

[0403] Methodologies to Isolate and Purify Clinical Grade Extracellular Vesicles for Medical Applications. Cells. 2022. 11.

[0404] 10.3390 / cellsl 1020186

[0405] FEUILLE DE REMPLACEMENT (RÈGLE 26)

[0036] , Clos-Sansalvador, M., Monguio-Tortajada, M., Roura, S., Franquesa, M., and Boiras, F. E. Commonly used methods for extracellular vesicles' enrichment: Implications in downstream analyses and use. Eur J Cell Biol. 2022. 101. 10.1016 / j.ejcb.2O22.151227

[0406]

[0037] , Liangsupree, T., Multia, E., and Riekkola, M. L. Modern isolation and separation techniques for extracellular vesicles. J Chromatogr A. 2021. 1636. 10.1016 / j. chroma.2020.461773

[0407]

[0038] , Brennan, K., Martin, K., FitzGerald, S. P., O’Sullivan, J., Wu, Y., Blanco,

[0408] A., Richardson, C., and Me Gee, M. M. A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum. Scientific Reports. 2020. 10. 10.1038 / S41598-020-57497-7

[0409]

[0039] , Panigrahi, A. R., Srinivas, L, and Panda, J. Exosomes: Insights and therapeutic applications in cancer. Transi Oncol. 2022. 21. 10.1016 / j. tranon.2022.101439

[0410]

[0040] , Xu, N., Shao, Y., Ye, K., Qu, Y., Memet, O., He, D., and Shen, J.

[0411] Mesenchymal stem cell-derived exosomes attenuate phosgene-induced acute lung injury in rats. Inhal Toxicol. 2019. 31.

[0412] 10.1080 / 08958378.2019.1597220

[0413]

[0041] , Thery, C., Amigorena, S., Raposo, G., and Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 2006. Chapter 3. 10.1002 / 0471143030. cb0322s30

[0414]

[0042] , Frescaline, G., Bouderlique, T., Mansoor, L., Carpentier, G., Baroukh, B.,

[0415] Sineriz, F., Trouillas, M., Saffar, J. L., Courty, J., Lataillade, J. J., Papy- Garcia, D., and Albanese, P. Glycosaminoglycan mimetic associated to human mesenchymal stem cell-based scaffolds inhibit ectopic bone formation, but induce angiogenesis in vivo. Tissue Eng Part A. 2013. 19. 10.1089 / ten.TEA.2012.0377

[0416]

[0043] , Jacquet-Guibon, S., Dupays, A. G., Coudry, V., Crevier-Denoix, N.,

[0417] Leroy, S., Sineriz, F., Chiappini, F., Barritault, D., and Denoix, J. M. Randomized controlled trial demonstrates the benefit of RGTA(R)

[0418] FEUILLE DE REMPLACEMENT (RÈGLE 26) based matrix therapy to treat tendinopathies in racing horses. PLoS One. 2018. 13. 10.1371 / journal. pone.0191796

[0419]

[0044] , US06689741. Cosmetic / dermatological composition

[0420]

[0045] , US2014301972A1. Use of biocompatible polymers for the preparation of a composition or a medical device

[0421]

[0046] , Holmes, B. B., DeVos, S. L, Kfoury, N., Li, M., Jacks, R., Yanamandra,

[0422] K., Ouidja, M. O., Brodsky, F. M., Marasa, J., Bagchi, D. P., Kotzbauer, P. T., Miller, T. M., Papy-Garcia, D., and Diamond, M. I. Heparan sulfate proteoglycans mediate internalization and propagation of specific proteopathic seeds. Proc Natl Acad Sci U S A. 2013. 110. 10.1073 / pnas.1301440110

[0423]

[0047] , Ouidja, M. O., Petit, E., Kerros, M. E., Ikeda, Y., Morin, C., Carpentier, G.,

[0424] Barritault, D., Brugere-Picoux, J., Deslys, J. P., Adjou, K., and Papy- Garcia, D. Structure-activity studies of heparan mimetic polyanions for anti-prion therapies. Biochem Biophys Res Commun. 2007. 363. 10.1016 / j.bbrc.2007.08.113

[0425]

[0048] , Cox, J., and Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b. -range mass accuracies and proteome-wide protein quantification. Nat Biotechnol. 2008. 26. 10.1038 / nbt.1511

[0426]

[0049] , Mine, S., Yamazaki, T., Miyata, T., Hara, S., and Kato, H. Structural mechanism for heparin-binding of the third Kunitz domain of human tissue factor pathway inhibitor. Biochemistry. 2002. 41.

[0427] 10.1021 / bi011299g

[0428]

[0050] , Chen, N., Chen, C. C., and Lau, L. F. Adhesion of human skin fibroblasts to Cyr61 is mediated through integrin alpha 6beta 1 and cell surface heparan sulfate proteoglycans. J Biol Chem. 2000. 275. 10.1074 / jbc.M003040200

[0429]

[0051] , Jones, G. C., and Riley, G. P. ADAMTS proteinases: a multi-domain, multi-functional family with roles in extracellular matrix turnover and arthritis. Arthritis Res Ther. 2005. 7. 10.1186 / ar1783

[0430]

[0052] , Jin, H., Xu, T., Chen, Q., Wu, C., Wang, P., Mao, Q., Zhang, S., Shen, J., and Tong, P. The Fate and Distribution of Autologous Bone Marrow

[0431] FEUILLE DE REMPLACEMENT (RÈGLE 26) Mesenchymal Stem Cells with Intra-Arterial Infusion in Osteonecrosis of the Femoral Head in Dogs. Stem Cells Int. 2016. 2016. 10.1155 / 2016 / 8616143

[0432]

[0053] , Jiang, T., Xia, G., Yang, B., Zhang, H. W., Yin, Y. S., Tang, C. W., and Yang, J. H. Application of Bone Marrow Mesenchymal Stem Cells

[0433] Effectively Eliminates Endotoxemia to Protect Rat from Acute Liver Failure Induced by Thioacetamide. Tissue Eng Regen Med. 2022. 19. 10.1007 / sl 3770-021 -00421 -5

[0434] FEUILLE DE REMPLACEMENT (RÈGLE 26)

Claims

Demands 1. Pharmaceutical composition comprising - a biocompatible polymer of the following general formula (I) AaXxYy (I) in which: A represents a monomer. X represents a grouping R1COOR2 OR -R9(C=0)RIO, Y represents an O or N-sulfonate group corresponding to one of the following formulas -R3OSO3R4, -RsNSOsRs, R7SO3R8 in which: Ri, R3, Rs, and R9 independently represent an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, and which may contain one or more aromatic rings, with the exception of benzylamine and benzylamine sulfonate. R2, R4, Re, and Rs independently represent a hydrogen atom or a cation M + , R? and R10 independently represent a bond, an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, a represents the number of monomers, x represents the degree of substitution of monomers A by groups X, y represents the degree of substitution of monomers A by groups Y, and - at least one extracellular vesicle.

2. Composition according to claim 1, wherein the identical or different monomers A are selected from sugars, esters, alcohols, amino acids, nucleotides, nucleic acids, proteins or derivatives thereof, preferably the monomers A are identical and are glucose. REPLACEMENT SHEET (RULE 26) 3. Composition according to claim 1 or 2, wherein the number of monomers "a" is such that the mass of said polymers of formula (I) is greater than or equal to 2000 daltons.

4. Composition according to any one of the preceding claims, wherein the substitution rate "x" is between 10 and 150%.

5. Composition according to any one of the preceding claims, wherein the substitution rate "y" is between 10 and 170%.

6. Composition for its use according to any one of the preceding claims, wherein said biocompatible polymer further comprises functional chemical groups Z, different from X and Y.

7. Composition according to claim 6, comprising a biocompatible polymer of general formula (II) following AaXxYyZz (II) in which: A represents a monomer. X represents a grouping R1COOR2 or -Rg(C=0)Rio, Y represents an O or N-sulfonate group corresponding to one of the following formulas -R3OSO3R4, -R5NSO3R6, R7SO3R8 in which: Ri, R3, Rs, and R9 independently represent an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, and which may contain one or more aromatic rings, with the exception of benzylamine and benzylamine sulfonate. R2, R4, Re, and Rs independently represent a hydrogen atom or a cation M + , R? and R10 independently represent a bond, an aliphatic hydrocarbon chain, possibly branched and / or unsaturated, REPLACEMENT SHEET (RULE 26) Z represents a group chosen from the group comprising amino acids, fatty acids, fatty alcohols, ceramides, or derivatives thereof, or even nucleotide addressing sequences. a represents the number of monomers, x represents the degree of substitution of monomers A by groups X, y represents the degree of substitution of monomers A by groups Y, z represents the degree of substitution of monomers A by groups Z, and - at least one extracellular vesicle.

8. Composition according to claim 6 or 7, wherein the substitution rate "z" of all monomers A by Z groups is from 1 to 50%.

9. Composition according to any one of claims 1 to 8, wherein said extracellular vesicle is an extracellular vesicle of eukaryotic and prokaryotic cells.

10. Composition according to any one of claims 1 to 9, wherein said extracellular vesicle is an extracellular vesicle of eukaryotic cells selected from the group comprising adult stem cells, native or induced pluripotent cells (iPS), multipotent or unipotent cells, and differentiated cells.

11. Composition according to claim 10, wherein the extracellular vesicle is selected from the group comprising exosomes and apoptotic bodies.

12. Composition according to any one of claims 1 to 11, wherein the extracellular vesicle further comprises at least one heparan-binding protein selected from the group comprising prmtl target chromatin (“chromatin target of prmtl” (Chtop)), the inhibitor REPLACEMENT SHEET (RULE 26) tissue factor pathway inhibitor (Tfpi), disintegrin and metalloproteinase with thrombospondin type 1 motif 4 (Adamts4), cysteine-rich angiogenic protein 61 (Ccn1), and neuron-derived neurotrophic factor (Ndnf).

13. Composition according to any one of claims 1 to 12 for its use in the prevention and / or treatment of tissue damage.

14. Composition for its use according to claim 13 wherein the tissue lesions are selected from the group comprising lesions of the musculoskeletal system, vascular lesions, cardiac lesions, lesions of the central nervous system, lesions of the peripheral nervous system, lesions of the sensory systems, lesions of the respiratory system, lesions of the digestive tract, lesions of bone tissue, lesions of pancreatic tissue, lesions of liver tissue, lesions of kidney tissue, lesions of the urinary tract and / or lesions of vaginal tissue.

15. A method for preparing extracellular vesicles with at least one biocompatible polymer as defined in any one of claims 1 to 14 comprising the steps of: a. culturing cells in a medium M1 b. culturing said at least one cell obtained in step a in a medium M2 comprising a biocompatible polymer as defined in any one of claims 1 to 14, c. Recovering the extracellular vesicles obtained in step b from the medium M2.

16. A preparation method according to claim 15, wherein the duration of step a) of incubation is from 1 minute to 72 hours REPLACEMENT SHEET (RULE 26) 17. A preparation method according to claim 15 or 16, wherein the medium M2 comprises a concentration of said biocompatible polymer from 1 picogram / mL to 10 mg / mL, preferably from 0.1 to 100 pg / mL.

18. Extracellular vesicle obtainable according to the process defined in any one of claims 15 to 17.

19. Extracellular vesicle according to claim 18 for its use in the prevention and / or treatment of tissue injury.

20. Extracellular vesicle for use according to claim 19, wherein the tissue lesion is selected from the group comprising a locomotor system lesion, a vascular lesion, a lymphatic system lesion, a cardiac lesion, a central nervous system lesion, a peripheral nervous system lesion, a sensory systems lesion, an upper and / or lower respiratory system lesion, a digestive tract lesion, a bone tissue lesion, a pancreatic tissue lesion, an ophthalmic system lesion, a liver tissue lesion, a renal tissue lesion, a skin tissue lesion, a urinary tract lesion and / or a vaginal tissue lesion and / or a uterine lesion and / or a penile lesion. REPLACEMENT SHEET (RULE 26)

Images