Method for producing a hydrogel comprising a cross-linked silylated polysaccharide

Abstract

The present invention relates to a method for producing a hydrogel comprising a cross-linked polysaccharide, in particular, a method for producing an injectable hydrogel comprising cross-linked hyaluronic acid. The present invention also relates to a hydrogel, preferably injectable, that can be obtained by the method, a composition containing the hydrogel, and the uses of said hydrogel.

Description

[0001] DESCRIPTION

[0002] TITLE: PROCESS FOR PREPARING A HYDROGEL COMPRISING A CROSSLINKED SILYLATED POLYSACCHARIDE

[0003] TECHNICAL FIELD

[0004] The present invention relates to a method for preparing a hydrogel comprising a crosslinked polysaccharide, in particular, to a method for preparing an injectable hydrogel comprising crosslinked hyaluronic acid. The present invention also relates to a hydrogel, preferably injectable, obtainable by the method, a composition comprising the hydrogel, and uses of this hydrogel.

[0005] STATE OF PRIOR ART

[0006] Polysaccharide hydrogels are used in various fields such as aesthetics, cosmetics, and therapy. They can, in particular, replace biological tissues. In particular, hyaluronic acid (HA) gels find applications in ophthalmology, periodontology, rheumatology, and cosmetic surgery. Hyaluronic acid hydrogels are used in particular to fill soft tissues, preferably skin, with volume defects such as wrinkles and scars, or to increase the volume of soft tissues.

[0007] To obtain hyaluronic acid gels with desirable mechanical properties, in vivo durability, and resistance to degradation for soft tissue filling, hyaluronic acid is generally crosslinked with one or more crosslinking agents. Conventional crosslinkers have at least two reactive functions with functional groups present on the polysaccharide that allow them to link polysaccharide molecules together and thus crosslink them. As a result, these crosslinkers exhibit some in vivo toxicity because their at least two reactive functions with functional groups present on the polysaccharide are also reactive with groups present on biopolymers and can allow them to also react with biopolymers such as peptides, carbohydrates, and DNA and thus crosslink them.

[0008] For reasons of biocompatibility and product safety, it is therefore desirable to reduce the quantities of crosslinking agent conventionally used in order to maintain the least modified polysaccharide possible. However, below a certain threshold, the prepared gels no longer exhibit suitable properties. In particular, hyaluronic acid gels crosslinked with 1,4-butanediol diglycidyl ether (BDDE) with a degree of modification of approximately 1% are very poorly cohesive.

[0009] To address this problem, various process parameter modifications have already been attempted.

[0010] In particular, it has already been proposed to modify the crosslinking medium by adding different alkali halide or phosphate salts, or by increasing the concentration of hyaluronic acid and / or NaOH (WC2014 / 064633, WO2016 / 096920, WC2017 / 016917).

[0011] Adjusting the duration and temperature of the crosslinking reaction has also been studied (Facile strategy involving low-temperature chemical cross-linking to enhance the physical and biological properties of hyaluronic acid hydrogel, Carbohydrate Polymers, 2018, Sukwha Kim) and some have succeeded in preparing hyaluronic acid, or chitosan, gels crosslinked with lower amounts of conventional crosslinking agent by freezing their reaction media (Preparation and physical properties of hyaluronic acid-based cryogels, Journal of Applied Polymer Science, 2015, Anna Strôm et al.; Chitosan gels and cryogels crosslinked with diglycidyl ethers of ethylene glycol and polyethylene glycol in acidic media, Biomacromolecules, 2019, Svetlana Bratskaya et al. and Hyaluronic acid cryogels with non-cytotoxic crosslinker genipin, Materials Letters, Joahanna Roether). However, the gels thus obtained are not homogeneous.

[0012] It is therefore still desirable to find a way to further reduce the amounts of conventional crosslinking agent used to obtain crosslinked polysaccharide gels, such as crosslinked hyaluronic acid, with mechanical properties suitable for filling soft tissues.

[0013] Furthermore, it has been considered to functionalize biopolymers, such as hyaluronic acid, with alkoxysilane groups capable of reacting with each other by sol-gel condensation reaction to form Si-O-Si bonds. The prepared gels are thus crosslinked without conventional crosslinking agents. This is notably illustrated in WO2011 / 089267 and WO2017 / 009200. However, this type of gels using only silicon derivatives are not very stable to heat sterilization and become solutions after treatment. Moreover, neither WO2011 / 089267 nor WO2017 / 009200 applies such a final sterilization step. Moreover, it should be noted that WO2011 / 089267 does not result in the formation of a gel with desirable mechanical properties for injection. Sol-gel condensation reactions are known to be favored at pH close to neutrality and / or in dehydrated media, particularly obtained by drying (Lee et al., One-pot synthesis of silane-modified hyaluronic acid hydrogels for effective antibacterial drug delivery via sol-gel stabilization, Colloids and surfaces B: Biotinterfaces, 2019, 174:308-315). However, these conditions have several drawbacks. Indeed, at pH close to neutrality, at room temperature, without drying, the probability of encounter between two silanol groups remains low and an insufficient number of Si-O-Si bonds are formed. During drying: heat-sensitive polymer chains, such as hyaluronic acid, degrade and generate low-molecular-weight polymer fragments whose biocompatibility is uncertain; the polymer concentration in the reaction medium is difficult to control; temperature gradients are created in the reaction medium, which is then inhomogeneous; bubble generation is possible; and drying of the final gel is possible.

[0014] There is therefore still a need to provide new hydrogels based on crosslinked polysaccharide, in particular crosslinked hyaluronic acid, containing lesser amounts of conventional crosslinking agent such as BDDE, or even containing no conventional crosslinking agent at all, and advantageously capable of being injected and / or sterilized.

[0015] SUMMARY

[0016] The present invention relates to a method for preparing a hydrogel, preferably injectable, comprising the following steps: a) providing at least one polysaccharide; b) providing at least one molecule of formula Chem. I: or a salt thereof, wherein:

[0017] T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide group, or an acid anhydride residue; A represents a chemical bond or a spacer group;

[0018] R 5 and R 6 , identical or different, represent a hydrogen atom; a halogen atom; a -OR group 4 with R 4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more group(s) chosen from a halogen atom, an aryl and a hydroxyl;

[0019] R 10represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; c) functionalization of the polysaccharide with at least one molecule of formula Chem.

[0020] I; d) crosslinking by sol-gel reaction of the functionalized polysaccharide to give a hydrogel; in which step d) comprises crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 9 and less than 14, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure P, for a duration t ranging from 2 weeks to 17 weeks, or in which step d) comprises crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 6.8 and less than or equal to 7.8,at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t of between 1 hour and 48 hours.,

[0021] The present invention also relates to a hydrogel obtainable by a method as described previously and below, as well as to a cosmetic or pharmaceutical composition comprising such a hydrogel.

[0022] Finally, the present invention relates to such a hydrogel or such a composition for its use in filling and / or replacing tissues; for preventing and / or treating the alteration of the viscoelastic or biomechanical properties of the skin; for filling volume defects of the skin, in particular for filling wrinkles, fine lines and scars; for reducing nasolabial folds and bitterness folds; for increasing the volume of the cheekbones, chin or lips; for restoring the volumes of the face, in particular the cheeks, temples, the oval of the face, and the area around the eyes; for reducing the appearance of wrinkles and fine lines; or for regenerating, moisturizing, firming or restoring the radiance of the skin, in particular by mesotherapy.

[0023] The invention also relates to the use of such a hydrogel or such a composition, the composition comprising at least one cosmetic active ingredient, for the modified, delayed or prolonged release of cosmetic active ingredients.

[0024] Other aspects of the invention are as described in the claims and hereinafter.

[0025] DEFINITIONS

[0026] The term "gel" refers to a polymer network that is expanded throughout its volume by a fluid. This means that a gel is formed of two media, one "solid" and the other "liquid", dispersed in each other. The so-called "solid" medium consists of long polymer molecules connected to each other by weak bonds (e.g. hydrogen bonds) or by covalent bonds (crosslinking). The liquid medium consists of a solvent. A gel generally corresponds to a product that has a phase angle 5 less than or equal to 45° at 1 Hz for a deformation of 0.1% or a pressure of 1 Pa, advantageously a phase angle 5 ranging from 2° to 45° or ranging from 20° to 45°.

[0027] The term "hydrogel" designates a gel as defined above in which the solvent constituting the liquid medium is predominantly water (for example at least 90%, in particular at least 95%, in particular at least 99% by weight of the liquid medium). Preferably, the liquid medium comprises, in particular consists of, a buffer solution, advantageously allowing a pH of the liquid medium of between 6.8 and 7.8, in particular a saline phosphate buffer.

[0028] The term “injectable gel” refers to a gel that can flow and be injected manually using a syringe equipped with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 30 G, 27 G, 26 G, 25 G hypodermic needle. Preferably, an “injectable gel” is a gel having an average extrusion force less than or equal to 25 N, preferably ranging from 5 to 25 N, more preferably ranging from 8 to 15 N, when measured with a dynamometer, at a fixed speed of approximately 12.5 mm / min, in syringes with an external diameter greater than or equal to 6.3 mm, with a needle with an external diameter less than or equal to 0.4 mm (27 G) and a length of 1”, at room temperature.

[0029] The "stringiness" of a product refers to its ability to be stretched between two surfaces to which it has adhered. The stringiness can be determined using a texturometer, sensory analysis carried out by a panel, or rheological and mechanical measurements including phase angle measurement (5) or tensile tests. In particular, this characteristic can be measured as described by P. Micheels et al. (Micheels et al., Comparison of two Swiss-designed hyaluronic acid gels: six-month clinical follow-up, Journal of Drug in Dermatology, 2017, 16:154-161, "Resistance to stretching") or by performing a Tack test and measuring the length of the gel threads in tension.

[0030] The term "polysaccharide" refers to a polymer composed of monosaccharides (preferably D-enantiomers) joined together by glycosidic bonds. The term "monosaccharide", also called "ose", refers to an unmodified or modified monosaccharide.

[0031] An “unmodified monosaccharide” means a compound of the formula H-(CHOH) X-CO- (CHOH)yH with x and y representing, independently of each other, an integer ranging from 0 to 5 provided that 2 < x+y < 5, the monosaccharide being able to be in a linear form represented by the above-mentioned formula or being able to be in a cyclized form by reaction of the CO function (aldehyde or ketone) with one of the OH groups to form a hemiacetal or hemiketal group. Preferably, the monosaccharide is in cyclized form. There are two types of ose: aldoses which carry an aldehyde function (when x or y is 0) and ketoses which carry a ketone function (when neither x nor y is 0). Monosaccharides are classified by number of carbons. For example, 6-carbon monosaccharides (x+y=5) are hexoses with the formula CeH^Oe and can be ai lose, altrose, glucose, mannose, gulose, idose, galactose or talose.The 5-carbon monosaccharides (x+y=4) are pentoses of formula C5H10O5 and may be ribose, arabinose, xylose, or lyxose. Preferably, the monosaccharide is a hexose, i.e. x+y = 5. A monosaccharide further comprises x+y asymmetric carbons and therefore 2. (x+y-1) pairs of enantiomers. Each pair of enantiomers is designated by a different name and the enantiomers of the same pair are called D and L enantiomers respectively.

[0032] A “modified monosaccharide” means an unmodified monosaccharide as defined above, including, for example:

[0033] - one or more of the OH functional groups have been replaced by another functional group, for example:

[0034] (i) a group OR with R representing a group (Ci-Ce)alkyl such as methyl or ethyl; hydroxy-(Ci-C6)alkyl such as hydroxyethyl (-CH2CH2OH) or hydroxypropyl (-CH2- CH(OH)-CH3); carboxy-(Ci-Ce)alkyl such as carboxymethyl (-CH2COOH); or CO-(Ci-Ce)alkyl such as acetyl; and / or

[0035] (ii) a group NR'R” with R' and R” representing, independently of each other, H, (C1-C6)alkyl or CO-(C1-C6)alkyl such as acetyl; and / or

[0036] (iii) an OSO3H group; and / or

[0037] - the terminal CH2OH function(s) have been replaced by a COOH or CHO group;

[0038] - a -CH(OH)-CH(OH)- bond is oxidized to give two terminal -CHO (aldehyde) groups in place of this bond; and / or

[0039] - a terminal CH2OH function was condensed with an OH functional group to form an -O-CH2- chain.

[0040] The term "repeating unit" of a polysaccharide refers to a structural unit consisting of one or more (usually 1 or 2) monosaccharides whose repetition produces the complete polysaccharide chain.

[0041] Some or all monosaccharides may be in a modified form. Monosaccharides, when modified, may be in different modified forms.

[0042] The term "physiologically acceptable" means that which is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for human or veterinary cosmetic (i.e., non-therapeutic) or therapeutic use, including use by injection into the human or animal body or for topical application to the skin.

[0043] The “salts” useful in the context of the present invention are preferably physiologically acceptable salts. The terms “physiologically acceptable salts” designate in particular:

[0044] 1) pharmaceutically acceptable acid addition salts formed with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like;or formed with pharmaceutically acceptable organic acids such as formic acid, acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-toluenesulfonic acid, trimethylacetic acid, trifluoroacetic acid and the like, and;

[0045] 2) pharmaceutically acceptable base addition salts formed when an acid proton present in the parent compound is either replaced by a metal ion, for example an alkali metal ion (eg Na, K), an alkaline earth metal ion (eg, Ca, Mg), a zinc ion, a silver ion or an aluminum ion; or coordinated with a pharmaceutically acceptable organic base such as diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like; or with a pharmaceutically acceptable inorganic base such as aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide and the like.

[0046] The “degree of modification” (MOD) of a polysaccharide, such as hyaluronic acid, corresponds to the molar quantity of modifying agent, such as the quantity of crosslinking agent and / or functionalizing agent linked to the polysaccharide, by one or more of its ends, expressed per 100 moles of repeating units of the polysaccharide. It can be determined by methods known to those skilled in the art such as Nuclear Magnetic Resonance (NMR) spectroscopy.

[0047] The “degree of functionalization” (DOF) corresponds to the molar quantity of functionalizing agent linked to the polysaccharide, by one end, expressed per 100 moles of repeating units of the polysaccharide. It can be determined by methods known to those skilled in the art such as Nuclear Magnetic Resonance (NMR) spectroscopy.

[0048] The “molar crosslinking rate” (TR), expressed in %, designates the molar ratio of the quantity of crosslinking agent to the quantity of repeating unit of the polysaccharide introduced into the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the crosslinking medium.

[0049] The “molar functionalization rate”, expressed in %, designates the molar ratio of the quantity of functionalizing agent relative to the quantity of repeating units of the polysaccharide used in the crosslinking reaction medium expressed per 100 moles of repeating units of the polysaccharide in the functionalization medium.

[0050] The term "molar modification rate" means the molar ratio of the amount of modifying agent (e.g., crosslinking agent and / or molecule of formula Chem. I as a functionalizing agent) to the amount of repeating unit of the polysaccharide used in the modification medium. The term "therapeutic active ingredient" means a substance for curing, alleviating the symptoms of and / or preventing a disease; a substance having curative or preventive properties with respect to human or animal diseases, as well as any substance that can be used in humans or animals or that can be administered to them, with a view to establishing a medical diagnosis or restoring, correcting or modifying their physiological functions by exerting a pharmacological, immunological or metabolic action.

[0051] The expression "cosmetic active ingredient" means any non-therapeutic substance intended in particular to be placed in contact with various superficial parts of the human body, such as the epidermis, hair and capillary systems, nails, lips, chest and teeth, with a view, exclusively or mainly, to cleaning them, protecting them, perfuming them, maintaining them in good condition, modifying their appearance or odor.

[0052] The term "approximately" means that the value concerned may be 10% lower or higher, in particular 5%, in particular 1%, than the indicated value.

[0053] An “aqueous reaction medium” means a reaction medium whose solvent is predominantly water (for example at least 90%, in particular at least 95%, notably at least 99% by weight of the total solvent) or even is water.

[0054] The term "spacer group" refers to a fragment comprising at least one atom intended to link together two chemical groups within the same molecule. Preferably, the spacer group contains at least one carbon atom.

[0055] The term "halogen" refers to an atom of fluorine, chlorine, bromine or iodine.

[0056] An "epoxide" group is an ethylene oxide residue linked to the rest of the molecule by one of its carbon atoms.

[0057] An "N-succinimidyloxycarbonyl" group is a group with the formula Chem. GR1 below: -sulfosuccinimidyloxycarbonyl" is a group with the formula Chem. A "halocarbonyl" group is a group of formula -CO-Hal with Hal representing a halogen, such as Cl or Br.

[0058] A “carbodiimide” group is a group comprising a -N=C=N- motif, and more particularly a group of formula -N=C=NR a with R arepresenting an aliphatic hydrocarbon group comprising from 1 to 20 carbon atoms, preferably a (C1-C6)alkyl group, one or more carbon atoms of which are optionally replaced by a heteroatom chosen from O, S and N, in particular N.

[0059] An “acid anhydride residue” is a group comprising a -C(O)-OC(O)- unit, and more particularly a monovalent cyclic group comprising the -C(O)-O-C(O)- unit, such as a saturated monovalent hydrocarbon monocyclic group comprising 5 to 10, in particular 5 or 6, carbon atoms of which three successive carbon atoms are replaced by C(O)-OC(O) and optionally of which one or more, in particular one, additional carbon atoms, preferably not consecutive to the three carbon atoms substituted by CO-O-CO, are each replaced by a heteroatom such as N, O or S, in particular N. The acid anhydride residue may correspond in particular to the following Chem. GR3 formula:

[0060] The acid anhydride residue may also be selected from a maleic anhydride residue or a succinic anhydride residue.

[0061] The expression “aliphatic hydrocarbon chain” or “aliphatic hydrocarbon group” designates a linear, branched and / or cyclic hydrocarbon group, saturated or unsaturated but not aromatic, advantageously comprising from 1 to 50, in particular from 1 to 20, for example from 1 to 12 or from 1 to 6 carbon atoms. These will in particular be alkyl groups.

[0062] The term "branched aliphatic hydrocarbon chain" specifically refers to a main aliphatic hydrocarbon chain comprising at least one secondary aliphatic hydrocarbon chain.

[0063] The term "star-shaped aliphatic hydrocarbon chain" means a branched aliphatic hydrocarbon chain comprising several secondary aliphatic hydrocarbon chains all starting from a single branch point.

[0064] The expression "C1-Cx alkyl" or "(Cl-Cx)alkyl" or "alkyl containing 1 to x carbon atoms" designates a saturated, linear or branched monovalent hydrocarbon group containing 1 to x carbon atoms, with x being an integer, such as for example a methyl, ethyl, isopropyl, tert-butyl, n-pentyl, cyclopropyl, cyclohexyl group, etc.

[0065] The expression "(Cl-Cx)alkylene" denotes a saturated, linear or branched, divalent hydrocarbon group containing from 1 to x carbon atoms, with x being an integer, such as, for example, a methane-1,1-diyl, ethane-1,1-diyl, ethane-1,2-diyl, propane-1,3-diyl, butane-1,4-diyl, butane-1,3-diyl, butane-1,2-diyl, pentane-1,5-diyl, hexane-1,6-diyl, hexane-1,5-diyl, heptane-1,7-diyl, octane-1,8-diyl, nonane-1,9-diyl, decane-1,10-diyl, etc. These include a methane-1,1-diyl or propane-1,3-diyl group.

[0066] The expression “hydroxy-(C1-Cx)alkyl” denotes a (Cl-Cx)alkyl group as defined above substituted by a hydroxyl group (OH) such as for example a hydroxyethyl (-CH2CH2OH) or a hydroxypropyl (e.g. -CH2-CH(OH)-CH3).

[0067] The expression “carboxy-(C1-Cx)alkyl” designates a (Cl-Cx)alkyl group as defined above substituted by a carboxyl group (COOH) such as for example a carboxymethyl (-CH2COOH).

[0068] The expression “aryl” denotes a monovalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more cycles, such as for example a phenyl or naphthyl group.

[0069] The term "arylene" means a divalent aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, comprising one or more rings, such as a phenylene group.

[0070] The expression “aryl-(C1-Cx)alkyl” designates an aryl group as defined above, linked to the rest of the molecule via a (Cl-Cx)alkyl chain as defined above with x an integer, such as for example the benzyl or phenylethyl group.

[0071] The term "polyvalent group" means a group that can form multiple covalent bonds with other groups in the same compound or in two different compounds. The bonds to the other groups can be formed from the same atom of the polyvalent group or from different atoms of the polyvalent group, and preferably from different atoms of the polyvalent group. In particular, the polyvalent group is a divalent group and can therefore form two covalent bonds with two other groups in the same compound or in two different compounds. The number of covalent bonds that can be formed is referred to as the "valence" of the polyvalent group.The expression "partly concomitant" as used in expressions of the type "steps b) and c) are partly concomitant" means that the two steps are carried out partly, at the same time, under the same reaction conditions, but that at least one of the two steps is initiated or completed under reaction conditions different from the common reaction conditions.

[0072] DETAILED DESCRIPTION OF THE INVENTION

[0073] To overcome the aforementioned drawbacks, the inventors propose functionalizing the polysaccharide using a molecule (herein referred to as a functionalizing agent or molecule of formula Chem. I) comprising a single function capable of reacting with a functional group of the polysaccharide and a silylated group capable of reacting with another silylated group via a sol-gel reaction so as to allow crosslinking of the polysaccharide and form a hydrogel. The crosslinking by sol-gel reaction is, at least partially, carried out at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P. The duration t of the crosslinking by sol-gel reaction at temperature T and pressure P is a function of the pH of the reaction medium.

[0074] The "sol-gel reaction" consists of forming Si-O-Si bonds from Si-OR groups with R representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing 1 to 6 carbon atoms. This reaction takes place as follows:

[0075] (i) if R is not a hydrogen atom, a step of hydrolysis of at least part of the Si-OR groups to give Si-OH groups; then

[0076] (ii) a step of condensation of the Si-OH groups two by two or of a Si-OH group with a Si-OR group to form Si-O-Si bonds.

[0077] In the present invention, the polysaccharide is functionalized by means of a molecule of formula Chem. I so as to become the bearer of Si-OR groups which will be able to react together and lead to a crosslinked polysaccharide.

[0078] The molecule of formula Chem. I comprising a single reactive function with respect to the polysaccharide and allowing crosslinking only via a sol-gel reaction, it does not present the toxicity of conventional crosslinking agents: the molecule of formula Chem. I cannot directly crosslink biological molecules (proteins, DNA, etc.).

[0079] Furthermore, the proposed method allows to prepare hydrogels based on crosslinked polysaccharide: - with superior viscoelastic properties compared to an identical composition but prepared at room temperature;

[0080] - with a particular texture visible to the naked eye and detectable by touch, namely, the gels according to the invention are stringy, cohesive;

[0081] - with adaptable properties, on demand, by simply passing it to a temperature higher than the eutectic point temperature of the reaction medium and lower than or equal to the freezing point temperature of the reaction medium measured at atmospheric pressure for more or less long periods. This adaptability is particularly advantageous in the context of the preparation of compositions for the modified, delayed or prolonged release of active substances, the active substance being pre-incorporated into the composition or added extemporaneously: the longer the composition is frozen, the greater the sol-gel reaction will be and the slower the release of the active substance will be. Thus, it is possible to adapt the duration and / or intensity of release of the product on demand:

[0082] - retaining desirable mechanical properties after sterilization;

[0083] - stable over time compared to the compositions of the prior art;

[0084] - with preserved polysaccharide chains and therefore comprising fewer low molecular weight polysaccharide fragments, including when applying a very basic pH during preparation. Analysis of the molecular weights of the polysaccharides in the gels according to the invention, for example by SEC-MALLS, can be used to characterize this phenomenon;

[0085] - with additional biological effects such as improving skin quality in animals, particularly humans;

[0086] - in a single step, the functionalization and the sol-gel reaction can be simultaneous;

[0087] - possibly without using conventional crosslinking agents.

[0088] The present invention therefore relates to a process for preparing a hydrogel, preferably injectable, comprising the following steps: a) providing at least one polysaccharide; b) providing at least one molecule of formula Chem. I:

[0089] R 5

[0090] T - A - Si - OR 10 p>6 or a salt thereof, in which:

[0091] T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide group, or an acid anhydride residue;

[0092] A represents a chemical bond or a spacer group;

[0093] R 5 and R 6 , identical or different, represent a hydrogen atom; a halogen atom; a -OR group 4 with R 4representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more group(s) chosen from a halogen atom, an aryl and a hydroxyl;

[0094] R 10 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; c) functionalization of the polysaccharide with at least one molecule of formula Chem.

[0095] I; d) crosslinking by sol-gel reaction of the functionalized polysaccharide to give a hydrogel; wherein step d) comprises crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 9 and less than 14, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure P, for a duration t ranging from 2 weeks to 17 weeks, or wherein step d) comprises crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 6.8 and less than or equal to 7.8,at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure P, for a duration t of between 1 hour and 48 hours. It should be understood from the above that step d) is carried out at least partially at pressure P and temperature T. The duration t of the crosslinking by sol-gel reaction at pressure P and temperature T depends on the pH of the reaction medium.,

[0096] In the above process, the polysaccharide(s) may be in salt form.

[0097] The present invention also relates to a hydrogel capable of being obtained by the method according to the invention.

[0098] The present invention also relates to a composition comprising a hydrogel according to the invention, as well as the therapeutic, cosmetic or aesthetic applications of the hydrogels or compositions according to the invention.

[0099] PROCESS

[0100] The steps of the method of the present invention may be as described below.

[0101] Step a)

[0102] Step a) of the method according to the invention consists of providing at least one polysaccharide. The polysaccharide may be in salt form.

[0103] Polysaccharide can be any polymer composed of monosaccharides joined together by glycosidic bonds.

[0104] Preferably, the polysaccharide is selected from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof.

[0105] "Pectin" is a polysaccharide composed of a skeleton of D-galacturonic acid in acid form, possibly esterified by methanol, and L-rhamnose capable of forming branches with other oses.

[0106] "Chitosan" and "chitin" are each a polysaccharide composed of repeating D-glucosamine units linked together in a B-(1,4) manner, a portion of which is N-acetylated. Chitosan specifically has an acetylation degree of less than 50%, while chitin specifically has an acetylation degree of greater than 50%. "Cellulose" is a polysaccharide composed of a linear chain of D-glucose molecules. "Cellulose derivatives" include methylcellulose, ethylcellulose, ethylmethylcellulose, hydroxypropylmethylcellulose (HPMC), hydroxyethylcellulose (HEC), hydroxypropylcellulose (HPC), and carboxymethylcellulose (CMC).

[0107] "Agarose" is a polysaccharide comprising as a repeating unit a disaccharide of D-galactose and 3,6-anhydro-L-galactopyranose.

[0108] Glycosaminoglycans are linear polysaccharides composed of repeating disaccharide units, said disaccharides containing a hexosamine (glucosamine (GIcN) or galactosamine (GaIN)) and another ose (glucuronic acid (GIcA), iduronic acid (IdoA) or galactose (Gai)). The hexosamine and the other ose may optionally be sulfated and / or acetylated. The glycosaminoglycan may be, in particular, hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin or chondroitin sulfate.

[0109] "Hyaluronic acid" is a glycosaminoglycan whose repeating unit is a disaccharide composed of D-glucuronic acid and N-acetyl-D-glucosamine, linked together by alternating glycosidic bonds |3-(1,4) and |3-(1,3). When hyaluronic acid is in the form of a salt, it is also referred to as "hyaluronate" or "hyaluronan". In the context of the present invention, hyaluronic acid may have a weight-average molecular mass ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, even more preferably greater than 0.05 MDa, for example ranging from 0.07 to 10 MDa or from 0.07 to 5 MDa, or from 0.5 to 5 MDa or from 1 to 5 MDa or from 2 to 4 MDa. Hyaluronic acid may be in salt form, particularly in physiologically acceptable salt form such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof.More specifically, hyaluronic acid is in acid form or in sodium salt form (NaHA).

[0110] "Heparosan" or "heparosan" is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid (GIcA) linked by an α-(1,4) bond to an N-acetyl glucosamine (GIcNAc). Each disaccharide repeating unit is linked to the next by a α-(1,4) bond.

[0111] Chondroitin sulfate is a glycosaminoglycan whose repeating unit is a disaccharide composed of glucuronic acid linked in p-(1,3) to sulfated N-acetyl galactosamine, i.e. it includes at least one sulfate substituent. Each disaccharide repeating unit is linked to the next by a P-(1,4) bond. Dermatan sulfate is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, i.e. it includes at least one sulfate substituent, of L-iduronic acid and N-acetyl-galactosamine linked by a(1-3) bonds. Advantageously, the disaccharide is sulfated at the C-4 position of N-acetyl-galactosamine, at the C-6 position of N-acetyl-galactosamine, at the C-2 position of L-iduronic acid, or at a combination of these positions. Each disaccharide repeat unit is connected to the next by a |3-(1,4) bond.

[0112] "Keratan sulfate" or "keratan sulfate" is a glycosaminoglycan whose repeating unit is a sulfated disaccharide, i.e. comprising at least one sulfate substituent, composed of D-galactose and N-acetylglucosamine linked by alternating P(1-4) and P(1-3) bonds.

[0113] The polysaccharide may be in the form of a salt, in particular in the form of a physiologically acceptable salt such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt and mixtures thereof, more particularly in the form of sodium or potassium salt.

[0114] Advantageously, the polysaccharide is a glycosaminoglycan or a salt thereof, preferably hyaluronic acid or a salt thereof, more preferably hyaluronic acid or one of its physiologically acceptable salts such as the sodium salt, the potassium salt, the zinc salt, the silver salt and mixtures thereof, even more preferably hyaluronic acid or its sodium salt.

[0115] The polysaccharide generally has a weight average molecular mass ranging from 0.03 to 10 MDa.

[0116] Preferably, if the polysaccharide is hyaluronic acid, it has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, even more preferably greater than 0.05 MDa, for example ranging from 0.07 to 10 MDa or from 0.07 to 5 MDa, or from 0.5 to 5 MDa or from 1 to 5 MDa or from 2 to 4 MDa.

[0117] The polysaccharide may be supplied in hydrated form, wholly or partially, or in dry form, such as powder or fiber.

[0118] In some embodiments, in step a), the polysaccharide is provided in dry form such as powder or fiber.

[0119] When the polysaccharide is provided in hydrated form, it is in the form of a non-crosslinked gel or a solution. In particular, when the polysaccharide is in hydrated form, it is an aqueous non-crosslinked gel or an aqueous solution. More particularly, the polysaccharide is mixed with water, optionally with the addition of a phosphate buffer or a supplemented phosphate buffer, i.e. possibly comprising additional components as defined in step f).

[0120] Step b)

[0121] Step b) of the method according to the invention consists of providing at least one molecule of formula Chem. I as presented above.

[0122] Preferably, in formula Chem. I, T represents an isocyanate, sulfhydryl, amino, epoxide, vinyl, formyl, or carbodiimide group, more preferably, T represents an epoxide or amino group, even more preferably T represents an epoxide group.

[0123] Preferably, in the formula Chem. I, A represents a spacer group, more preferably a divalent aliphatic hydrocarbon chain, in particular linear or branched and saturated, comprising from 1 to 12 carbon atoms:

[0124] - in which are optionally intercalated, between two carbon atoms of said chain, one or more (in particular 1, 2, 3 or 4) divalent units chosen from arylenes, -O-, -S-, -S(O)-, -C(=O)-, -SO2- and -N(R 9 )- with R 9representing a hydrogen atom, an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl,

[0125] - said chain being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl.

[0126] Advantageously, A is a divalent aliphatic hydrocarbon chain, in particular linear or branched and saturated, in which are optionally intercalated, between two carbon atoms of said chain, one or more divalent units -O-, more advantageously from 1 to 4 divalent units -O-, even more advantageously a divalent unit O.

[0127] Preferably, A is a (C1-C12)alkylene chain in which are optionally intercalated, between two carbon atoms of said chain, one or more divalent -O- units, more preferably from 1 to 4 divalent -O- units, even more preferably one divalent -O- unit.

[0128] In particular, A represents a divalent chain -(C1-C6)alkylene-O-(C1-C6)alkylene-, in particular -(C1-C4)alkylene-O-(C1-C4)alkylene-, more particularly a divalent chain -CH2-O-(CH2)3-, the CH2 group being linked to T and the (CH2)3 group being linked to Si in the molecule of formula Chem. I. Advantageously, the spacer group will also make it possible to avoid steric hindrance between the silylated group and the T group of the molecule of formula Chem. I, while ensuring a stable bond between these two groups.

[0129] Preferably, in the formula Chem. I, R 5 and R 6 , identical or different, represent a -OR group4 with R 4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl and a hydroxyl.

[0130] In particular, R 5 and R 6 , identical or different, represent a -OR group 4 with R 4 representing a (C1-C6)alkyl group; or a (C1-C6)alkyl group.

[0131] Advantageously, R 5 and R 6 , identical or different, represent a -OR group 4 with R 4 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably with R 4representing an aliphatic hydrocarbon group having from 1 to 6 carbon atoms, such as a (C1-C6)alkyl group.

[0132] Preferably, in the formula Chem. I, R 10 represents a hydrogen atom or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms such as a (C1-C6)alkyl group, more advantageously R 10 represents an aliphatic hydrocarbon group having from 1 to 6 carbon atoms such as a (C1-C6)alkyl group.

[0133] Preferably, the molecule of formula Chem. I is such that:

[0134] - T is as defined above and advantageously represents an amino or epoxide group, preferably an epoxide group;

[0135] - A is a divalent chain -(C1-C6)alkylene-O-((C1-C6)alkylene-, in particular -(C1-C4)alkylene-O-(C1-C4)alkylene-, such as -CH2-O-(CH2)3-, the CH2 group preferably being linked to T and the (CH2)3 group being linked to Si in the molecule of formula Chem. I;

[0136] - R 5 and R 6 , identical or different, are each a -OR grouping 4 with R 4 representing a (C1-C6)alkyl group, preferably a methyl or an ethyl; or a (C1-C6)alkyl group, preferably a methyl or an ethyl; and

[0137] - R 10 is a (C1-C6)alkyl group, preferably methyl or ethyl; the R groups 5 , R 6 and GOLD 10 which may be identical.

[0138] In particular, the molecule of formula Chem. I is chosen from (3-aminopropyl)triethoxysilane (APTES), (3-glycidyloxypropyl)trimethoxysilane (GPTMS), 3-Glycidoxypropyldimethoxymethylsilane, 3-Glycidoxypropyldimethylethoxysilane, (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane, and mixtures thereof; preferably from (3-glycidyloxypropyl)trimethoxysilane (GPTMS), (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane, and mixtures thereof.

[0139] Step c)

[0140] The polysaccharide is functionalized with at least one molecule of formula Chem. I as presented above.

[0141] Step c) allows the polysaccharide chains to be functionalized. The functional group T of the molecule of formula Chem. I reacts with a functional group present on the polysaccharides so as to functionalize the polysaccharide chains. In particular, the functional group T of the molecule Chem. I thus reacts with an -OH or -COOH group, or a CHO function, present on polysaccharides such as hyaluronic acid. This produces functionalized polysaccharides comprising pendant bonds on a polysaccharide chain, said pendant bonds comprising a group -A-Si(R 5 )(R 6 )GOLD 10 , the group -A- Si(R 5 )(R 6 )GOLD 10 originating from the molecule of formula Chem. I from step b) which can provide biological properties to the hydrogel.

[0142] Preferably, in step c), the polysaccharide is functionalized in the presence of 0.01 to 0.50, preferably 0.05 to 0.45, in particular 0.10 to 0.25 moles of molecule of formula Chem. I or a salt thereof per 1 mole of repeating unit of the polysaccharide. Typically, the higher the weight-average molecular mass Mw of the polysaccharide, the lower the degree of functionalization of the molecule of formula Chem. I will be in order to obtain a hydrogel having equivalent mechanical properties, in particular similar viscoelastic properties (in particular elastic modulus G', stress at the intersection of G' and G” and / or phase angle 5). In other words, the higher the weight-average molecular mass Mw of the polysaccharide, the lower the molar quantity of molecule of formula Chem. I introduced in step c) will be.

[0143] The functionalization of the polysaccharide is typically carried out in an aqueous reaction medium.

[0144] In certain embodiments, in particular when T is an epoxide, the functionalization is carried out at a pH greater than or equal to 9, or greater than or equal to 10, more advantageously greater than or equal to 12, and in particular at a pH less than 14, for example less than or equal to 13.5. For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably sodium or potassium hydroxide. Advantageously, the reaction medium comprises sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0145] In certain embodiments, in particular when T is an amino group, the functionalization is carried out at a pH of less than 7, more advantageously greater than or equal to 4.5 and less than 7 or less than or equal to 6.5. For this, the reaction medium preferably comprises a Bronsted acid, more preferably hydrochloric acid, sulfuric acid, or acetic acid.

[0146] The mass concentration of polysaccharide in the functionalization reaction medium is advantageously between 50 and 300 mg / g of functionalization medium, preferably between 100 and 200 mg / g.

[0147] In certain embodiments, the functionalization of the polysaccharide is carried out at a temperature between 4°C and 60°C, more preferably between 10°C and 50°C, even more preferably between 10°C and 25°C. In these embodiments, the duration of the functionalization reaction can vary from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, even more particularly from 3 hours to 96 hours, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours.In certain embodiments, in particular when the functionalization and the crosslinking of the polysaccharide are concomitant or partly concomitant, the functionalization of the polysaccharide can be, at least in part, carried out at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at the pressure P and less than the temperature of the freezing point of the reaction medium as measured at the pressure P.

[0148] The higher the functionalization temperature, the shorter the functionalization time can be to obtain the same degree of functionalization.

[0149] Step d)

[0150] The functionalized polysaccharide is crosslinked by sol-gel reaction to give a hydrogel.

[0151] This step allows the polysaccharide chains to be crosslinked together when they are functionalized with molecules of formula Chem. I. Indeed, during this step, at least part of the Si-OR groups 10 and optionally at least part of the SiOR groups 4 will react two by two, possibly after hydrolysis of these groups, to form Si-O-Si bonds. This implies that two molecules of formula Chem. I grafted onto polysaccharide chains will react together via their Si-OR terminal groups 10 (or even SiOR 4 where applicable) and bind covalently via the formation of Si-O-Si bonds, thus allowing the polysaccharide chains to be bound together and crosslinked.

[0152] Crosslinked polysaccharides are thus obtained comprising crosslinking links between two polysaccharide chains, said crosslinking links comprising a divalent group -Si-O-Si-.

[0153] Therefore, step d) cannot take place before step c).

[0154] Step d) is carried out at least partially (i.e. in part or in full), at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P. The duration of maintaining these conditions depends on the pH of the reaction medium. Thus, when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14, the pressure P and the temperature T are maintained for a duration t ranging from 2 to 17 weeks, in particular from 2 to 10 weeks or from 2 to 9 weeks, for example from 3 to 6 weeks, more advantageously from approximately 4 to 5 weeks.When the pH of the reaction medium is greater than or equal to 6.8 and less than or equal to 7.8, the pressure P and the temperature T are maintained for a duration t ranging from 1 to 48 hours, preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours.

[0155] It should be understood that the reaction conditions (pH, T, P) set out above may correspond to the conditions applied throughout the duration of the crosslinking step (step d)) or may correspond to the conditions applied only during part of the duration of step d). In other words, the duration of the crosslinking step d) may be greater than the durations t indicated above, the reaction conditions (pH or T or P) applied in the additional time then being different from those set out above.

[0156] The freezing point temperature of the reaction medium refers to the temperature at which the mixture of the components of the reaction medium, on a macroscopic scale, solidifies, i.e. it becomes non-fluid. Below the freezing point, the mixture is in a frozen state characterized by the coexistence of components in solid and liquid form. The frozen state is maintained up to the eutectic point temperature of the reaction medium.

[0157] The eutectic point temperature of the reaction medium refers to the temperature below which the mixture of components of the reaction medium passes from a frozen state (coexistence of liquid and solid phases) to a completely solid state, i.e. a state in which all the components of the mixture are in solid form.

[0158] The freezing point and eutectic point of a mixture depend on the pressure to which the mixture is subjected, therefore the freezing point and eutectic point are measured at pressure P.

[0159] The freezing point and eutectic point can be determined by differential scanning calorimetry. This method allows phase transitions to be determined. To do this, the product to be studied is gradually cooled until its phase transitions are observed.

[0160] "Atmospheric pressure" is the pressure exerted by the air constituting the atmosphere on any surface in contact with it. It varies according to altitude. At an altitude of 0 m, the average atmospheric pressure is 101,325 Pa.

[0161] At temperature T and pressure P, the reaction medium is thus in frozen form.

[0162] The sol-gel reaction typically takes place in an aqueous reaction medium.

[0163] The mass concentration of polysaccharide in the reaction medium is advantageously between 50 and 300 mg / g of sol-gel reaction medium, preferably between 100 and 200 mg / g.

[0164] Preferably, the pressure P is between 10' 3 mbar and atmospheric pressure, more preferably, pressure P is atmospheric pressure.

[0165] Preferably, the temperature T is greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C, in particular from -30°C to -10°C or from -25°C to -15°C. Even more preferably, the temperature T is approximately -20°C.

[0166] Preferably, the pressure P is atmospheric pressure and the temperature T is greater than or equal to -55°C and less than or equal to -5°C, preferably it ranges from -35°C to -10°C, in particular from -30°C to -10°C or from -25°C to -15°C, even more preferably, the temperature T is approximately -20°C.

[0167] Advantageously, the reaction medium is placed and maintained at temperature T by contact of the container comprising the reaction medium with air or a liquid L at temperature T. The liquid L may in particular be ethylene glycol, glycerol or an azeotropic mixture of these with water. The liquid L will be chosen according to the desired temperature T so as to be liquid at this temperature T. More advantageously, the reaction medium is left at temperature T by contact of the container comprising the reaction medium with air at temperature T.

[0168] Typically, the lower the temperature T, the longer the time t is required to obtain hydrogels with similar mechanical properties. Indeed, the lower the temperature T, the lower the kinetics of the sol-gel reaction.

[0169] Similarly, the lower the functionalization rate, the longer the time t is to obtain hydrogels with similar mechanical properties.

[0170] In other words, the lower the molar quantity of molecule of formula Chem. I or a salt thereof per 1 mole of repeating unit of the polysaccharide, the fewer SiOH functions there are in the reaction medium and the lower the probability that 2 groups meet and react together, thus the longer the duration t must be to allow the Si-OH functions to react with each other and form crosslinks, and thus obtain a gel with desirable properties.

[0171] Thus, with a functionalization rate of 4 to 18%, the duration t is generally between 2 weeks and 15 weeks when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14.

[0172] With a functionalization rate of 4 to 8%, the duration t is generally between 8 weeks and 15 weeks when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14.

[0173] With a functionalization rate of 8 to 12%, the duration t is generally between 3 weeks and 10 weeks when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14.

[0174] With a functionalization rate of 12 to 18%, the duration t is generally between 2 weeks and 6 weeks when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14.

[0175] For the same molar quantity of molecule of formula Chem. I or a salt thereof per 1 mole of repeating unit of the polysaccharide, the lower the weight-average molecular mass Mw of the polysaccharide, the longer the time t is required to obtain hydrogels having similar mechanical properties. When the crosslinking by sol-gel reaction at temperature T and pressure P is carried out in a reaction medium at a pH greater than or equal to 9 or greater than or equal to 10 and less than 14, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably sodium or potassium hydroxide. Advantageously, the reaction medium comprises sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0176] Preferably, at the end of the duration t (crosslinking carried out at pressure P and temperature T), the pH of the reaction medium is adjusted to a physiological pH, preferably to a pH of approximately 6.8 to 7.8. It should be understood that at the end of the duration t, the reaction medium is brought back to room temperature and placed or maintained at atmospheric pressure (if P is equal to atmospheric pressure).

[0177] When the crosslinking by sol-gel reaction at temperature T and pressure P is carried out in a reaction medium at physiological pH (pH greater than or equal to 6.8 and less than or equal to 7.8) and the functionalization is carried out in a basic medium (pH greater than or equal to 9, or greater than or equal to 10, and less than 14), the pH of the reaction medium will be brought to a physiological pH before the temperature is brought to temperature T and before the pressure is brought to pressure P if the latter is lower than atmospheric pressure. In this case, the process will advantageously comprise a step of neutralizing the gel to reach this physiological pH, before the reaction medium is brought to temperature T and pressure P. For this, a Bronsted acid is preferably added to the reaction medium, preferably an aqueous solution of hydrochloric acid, an aqueous solution of sulfuric acid or an aqueous solution of acetic acid.

[0178] When the crosslinking by sol-gel reaction at temperature T and pressure P is carried out in a reaction medium at physiological pH (pH greater than or equal to 6.8 and less than or equal to 7.8) and the functionalization is carried out at a pH lower than 7, for example greater than or equal to 4.5 and less than 7 or less than or equal to 6.5, the pH of the reaction medium will be brought to a physiological pH before the temperature is brought to temperature T and before the pressure is brought to pressure P if the latter is lower than atmospheric pressure. Steps c) and d) concomitant or partly concomitant

[0179] Very generally, the process of the present invention comprises the concomitant or partly concomitant carrying out of steps c) and d). Concomitant carrying out of steps c) and d) makes it possible to shorten the duration of the process for preparing the hydrogel and to simplify it.

[0180] The method of the present invention then comprises steps a) to d) as described above and is characterized in that steps c) and d) are concomitant or partly concomitant. The method then comprises:

[0181] - functionalization and crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 9, or greater than or equal to 10, and less than 14, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t ranging from 2 weeks to 17 weeks, in particular from 2 to 10 weeks or from 2 to 9 weeks, for example from 3 to 6 weeks, more advantageously from approximately 4 to 5 weeks, or

[0182] - functionalization and crosslinking by sol-gel reaction carried out in a reaction medium at a pH greater than or equal to 6.8 and less than or equal to 7.8, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t of between 1 hour and 48 hours, preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours.

[0183] It should be understood from the above that steps c) and d) are at least partially (in part or in full) carried out under the conditions set out above (T, P, pH, t).

[0184] In some embodiments, the functionalization of the polysaccharide and the crosslinking of the functionalized polysaccharide are then carried out in the following manner:

[0185] 1) preparation of a reaction medium comprising the polysaccharide(s), the molecule(s) of formula Chem.l and a solvent, the pH of the reaction medium being greater than or equal to 9, or greater than or equal to 10, and less than 14; 2) optionally placing the reaction medium at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, even more preferably from 10 to 25°C, typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours;

[0186] 3) optionally adjusting the pH of the reaction medium to a pH greater than or equal to 6.8 and less than or equal to 7.8;

[0187] 4) placement of the reaction medium: at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t ranging from 2 weeks to 17 weeks, when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14, in particular from 2 to 10 weeks or from 2 to 9 weeks, for example from 3 to 6 weeks, more advantageously from approximately 4 to 5 weeks, or at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t of between 1 hour and 48 hours,preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours, when the pH of the reaction medium is greater than or equal to 6.8 and less than or equal to 7.8.,

[0188] In other words, the method of the present invention can then be defined as follows: a) providing at least one polysaccharide; b) providing at least one molecule of formula Chem. I as described above; c) functionalizing the polysaccharide with at least one molecule of formula Chem. I as described above; d) crosslinking by sol-gel reaction of the functionalized polysaccharide to give a hydrogel; wherein the functionalization and crosslinking of the functionalized polysaccharide are carried out in the following manner: 1) preparing a reaction medium comprising the polysaccharide(s), the molecule(s) of formula Chem. I and a solvent, the pH of the reaction medium being greater than or equal to 9, or greater than or equal to 10, and less than 14;

[0189] 2) optionally placing the reaction medium at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, even more preferably from 10 to 25°C, typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours;

[0190] 3) optionally adjusting the pH of the reaction medium to a pH greater than or equal to 6.8 and less than or equal to 7.8;

[0191] 4) placement of the reaction medium: at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t ranging from 2 weeks to 17 weeks, in particular from 2 to 10 weeks or from 2 to 9 weeks, for example from 3 to 6 weeks, more advantageously from approximately 4 to 5 weeks, when the pH of the reaction medium is greater than or equal to 9, or greater than or equal to 10, and less than 14, or at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the temperature of the eutectic point of the reaction medium as measured at pressure P and less than the temperature of the freezing point of the reaction medium as measured at pressure P, for a duration t of between 1 h and 48 hours,preferably greater than or equal to 6 hours and less than or equal to 36 hours, in particular greater than or equal to 7 hours and less than or equal to 36 hours, when the pH of the reaction medium is greater than or equal to 6.8 and less than 7.8.,

[0192] The solvent is typically water or a mixture comprising water and an organic solvent (for example an alcohol, in particular ethanol, or DMSO; typically a mixture comprising at least 90% by weight of water, or at least 95% or at least 99% by weight of water relative to the total weight of the solvent). The reaction medium typically comprises from 0.01 to 0.50, preferably from 0.05 to 0.45, in particular from 0.10 to 0.25 moles of molecule of formula Chem. I or a salt thereof per 1 mole of repeating unit of the polysaccharide.

[0193] The mass concentration of polysaccharide in the reaction medium is advantageously between 50 and 300 mg / g of solvent, preferably between 100 and 200 mg / g.

[0194] In certain embodiments, the functionalization and crosslinking are carried out in a reaction medium whose pH is greater than or equal to 9, or greater than or equal to 10, and less than 14. For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably sodium or potassium hydroxide. Advantageously, the reaction medium comprises sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0195] In these embodiments, according to a first variant, the reaction medium prepared in step 1) can be placed at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, even more preferably from 10°C to 25°C, typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours (step 2) before being placed at temperature T and pressure P for a period t (step 4). During step 2, the polysaccharide will be functionalized and a portion of the Si-OR groups 10 and optionally Si-OR groups 4 will condense together (pre-condensation), more Si-OR groups 10 and optionally Si-OR groups 4 condensing during step 4) (advanced condensation).

[0196] In these embodiments, according to a second variant, the reaction medium prepared in step 1) can be placed directly at the end of step 1) at temperature T and pressure P for a duration t (step 4).

[0197] At the end of time t (first and second variant), the temperature of the reaction medium is typically returned to room temperature and the reaction medium is placed at atmospheric pressure if the pressure P is different from atmospheric pressure. The pH of the reaction medium is then preferably brought to a physiological pH (approximately 6.8 to 7.8). For this, a Bronsted acid is preferably added to the reaction medium, preferably an aqueous hydrochloric acid solution, an aqueous sulfuric acid solution or an aqueous acetic acid solution. In certain embodiments, the crosslinking is carried out partially in a reaction medium whose pH is greater than or equal to 6.8 and less than 7.8. In these embodiments, a reaction medium having a pH greater than or equal to 9, or greater than or equal to 10, and less than 14 and comprising the polysaccharide(s), the molecule(s) of formula Chem.l and a solvent is prepared (step 1)). The reaction medium is placed at a temperature ranging from 4°C to 60°C, preferably from 10°C to 50°C, even more preferably from 10°C to 25°C typically for a period ranging from 1 hour to 2 weeks, more particularly from 3 hours to 1 week, for example from 3 hours to 80 hours, in particular from 3 hours to 75 hours (step 2). Then, the pH of the reaction medium is brought to a physiological pH (step 3)) before the temperature is brought to T and before the pressure is brought to pressure P if the latter is lower than atmospheric pressure. In this case, the process will advantageously comprise a step of neutralizing the gel to reach this physiological pH, before the reaction medium is brought to temperature T and pressure P.For this, a Bronsted acid is preferably added to the reaction medium, preferably an aqueous hydrochloric acid solution, an aqueous sulfuric acid solution or an aqueous acetic acid solution. The reaction medium is then placed at temperature T and pressure P for a duration t of between 1 h and 48 hours (step 4).

[0198] Preferably, the process of the present invention comprises only a single step of placing the reaction medium at temperature T and pressure P.

[0199] Step e)

[0200] The method according to the invention may comprise an additional step e) of crosslinking the polysaccharide with a conventional crosslinking agent, and more particularly the crosslinking of the polysaccharide provided in step a) or of the crosslinked polysaccharide obtained following step d), in the presence of at least one crosslinking agent or a salt thereof, said crosslinking agent comprising at least two functional groups Z as described below.

[0201] The “crosslinking agent” or “crosslinking agent” is a compound comprising at least two functional groups capable of binding covalently with functional groups present on the polysaccharide, such as OH, CHO, NH2 or COOH groups carried by the polysaccharide, and thus inducing bonds between the polysaccharide chains (crosslinking) and / or bonds on the same polysaccharide chain.

[0202] The crosslinking agent useful in the context of the present invention comprises at least two, preferably from 2 to 8, in particular 2, functional groups (designated "Z group") preferably chosen independently from the isocyanate (-N=C=O), amino (-NH2), epoxide, carboxyl (-COOH), N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate (-N=C=S), vinyl (-CH=CH2), formyl (-CH=O), hydroxyl (-OH), sulfhydryl (-SH), hydrazino (-NH-NH2), acylhydrazino (-CO-NH-NH2), aminoxy (-O-NH2), carbodiimide groups, and an acid anhydride residue. Preferably, the functional groups are identical.

[0203] The isocyanate group can react with an OH or NH2 group of the polysaccharide to form a carbamate or urea function. The amino group can react with a COOH group of the polysaccharide to form an amide function. The epoxide group can react with an OH or COOH group of the polysaccharide to form an ether or ester function. The carboxyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The N-succinimidyloxycarbonyl and N-sulfosuccinimidyloxycarbonyl groups can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The halocarbonyl group can react with an OH or NH2 group of the polysaccharide to form an ester or amide function. The isothiocyanate group can react with an OH or NH2 group of the polysaccharide to form a thiocarbamate or thiourea function.The vinyl group can react with an OH group of the polysaccharide to form an ether function. The formyl group can react with an OH or NH2 group of the polysaccharide to form a hemiacetal or hemiaminal function. The hydroxyl group can react with a COOH group of the polysaccharide to form an ester function. The sulfhydryl group can react with a COOH group of the polysaccharide to form a thioester function. The hydrazino group (-NH-NH2) can react with a CHO group of the polysaccharide to form a hydrazone function. The acylhydrazino group can react with a CHO group of the polysaccharide to form a carbonylated hydrazone function =NNHC(O)-. The aminoxy group can react with a CHO group of the polysaccharide to form an oxime function =NO-. The carbodiimide group can react with a COOH group of the polysaccharide to give a CO-NR function. a- CO-NH, and an acid anhydride residue can react with an OH or NH2 group of the polysaccharide to form an ester or amide function.

[0204] In particular, the T group of the molecule of formula Chem. I and the Z groups are identical.

[0205] Preferably, the functional groups Z are identical and represent an epoxide or vinyl group, more preferably epoxide.

[0206] According to another advantageous embodiment, the functional groups Z are identical and chosen from amino, vinyl, formyl, and carbodiimide groups, preferably are amino groups.

[0207] In particular, the crosslinking agent is selected from hexamethylene diisocyanate, 4,4'-diphenylmethylene diisocyanate, PEG20K-4-arm isocyanate, spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), poly(ethylene glycol) diamine, ethylenediamine, 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1 ,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end with a diglycidyl ether (CAS number: 130167-23-6), poly(ethylene glycol) diacid, disuccinimidyl suberate, bis(sulfosuccinimidyl)suberate, sebacoyl chloride, 1,4-butane diisothiocyanate, divinylsulfone (DVS), glutaraldehyde, polyethylene glycol, 1,5-pentanedithiol, adipic acid dihydrazide,bis-aminooxy-poly(ethylene glycol), diethylenetriaminepentaacetic acid dianhydride, and mixtures thereof.,

[0208] When the functional groups Z are epoxide groups, the crosslinking agent is preferably chosen from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), 1,3-bis(3-glycidyloxypropyl)tetramethyldisiloxane, poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6), hydroxyapatite beads modified to carry epoxy groups and mixtures thereof.

[0209] More preferably, the crosslinking agent is chosen from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDGE), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE), and mixtures thereof.

[0210] When the functional groups Z are amino groups, the crosslinking agent is preferably a polyamine chosen from spermine (or 1,12-diamino-5,9-diazadodecane), spermidine (or 1,8-diamino-5-azaoctane), cadaverine (or 1,5-diaminopentane), putrescine (or 1,4-diaminobutane), their salts or a mixture thereof, more preferably the crosslinking agent is a polyamine chosen from spermine, spermidine, their salts and their mixtures.

[0211] When the functional groups Z are amino groups, the crosslinking reaction of step e) with the polysaccharide is advantageously carried out in the presence of at least one activator, and where appropriate combined with at least one coupling auxiliary.

[0212] In this regard, the activator may be selected from water-soluble carbodiimides such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl-3-[3-(trimethylamino)propyl]carbodiimide hydrochloride (ETC), 1-cyclohexyl-3-(2-morphilinoethyl)carbodiimide (CMC), their salts and mixtures thereof, preferably is represented by EDC.

[0213] With respect to the coupling aid, when present, it may be selected from N-hydroxy succinimide (NHS), N-hydroxybenzotriazole (HOBt), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazole (HOOBt), 1-hydroxy-7-7azabenzotriazole (HAt) and N-hydroxysylfosuccinimide (sulfo NHS), and mixtures thereof, preferably is represented by HOBt.

[0214] The crosslinking agent may be selected from hydroxyapatite beads modified to carry epoxy groups, a compound of formula Chem. Il as described below, and mixtures thereof.

[0215] Preferably, the crosslinking agent is a compound of formula Chem. It:

[0216] Y-(Z) n in which the groups Z, identical or different, are as defined above, n is an integer greater than or equal to 2, in particular ranging from 2 to 8, preferably equal to 2,

[0217] Y is a polyvalent hydrocarbon group, notably aliphatic, having a valence of n and containing from 1 to 150 carbon atoms:

[0218] - in which one or more (for example 1 to 150, or 1 to 50 or 1 to 15 or 1 or 2) CH2 units are optionally replaced by one or more divalent units chosen from arylenes; -O-; -S-; -S(O)-; -C(=O)-; -SO2-; -N(R 1 )- ; and -[SiR 2 R 3 O] m -SiR 2 R 3 - with

[0219] R 1representing a hydrogen atom, an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl; m an integer between 1 and 20; and

[0220] R 2 and the Rs 3 , identical or different, representing a hydrogen atom; a halogen atom; a -OR group 11 with R 11 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl,

[0221] - said polyvalent group being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, and an aryl-(C1-C6)alkyl, preferably unsubstituted.

[0222] In particular n is an integer ranging from 2 to 8, preferably n represents 2, 3 or 4, even more preferably n is equal to 2.

[0223] Advantageously, R 1 represents a hydrogen atom or a (C1-C6)alkyl group.

[0224] In particular, R 2 and the Rs 3 , identical or different, represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0225] Preferably, in the definition of Y, the polyvalent hydrocarbon group may be a polyvalent aliphatic or aromatic hydrocarbon group, preferably aliphatic and in particular saturated, having a valence of n and comprising from 1 to 150 carbon atoms, preferably from 1 to 50 carbon atoms, more preferably from 1 to 20 carbon atoms, even more preferably from 2 to 20 carbon atoms.

[0226] In particular, in the definition of Y, the polyvalent hydrocarbon group is a polyvalent aliphatic, saturated, in particular linear, hydrocarbon group.

[0227] Preferably, Y is a polyvalent hydrocarbon group as described above in which one or more CH2 units are optionally replaced by one or more divalent units selected from -O-, -SO2-, -[SiR 2 R 3 O] m -SiR 2 R 3 - and -NH-, with R 2 , R 3 and m as described above.

[0228] In particular, Y is a polyvalent hydrocarbon group as described above, preferably aliphatic and saturated, and in particular linear, branched, or star-shaped, and optionally in which:

[0229] - at least two CH2 units are replaced by -O-, particularly between 1 and 50 CH2 units, more particularly between 1 and 15 CH2 units, or - at least one, preferably one or two, CH2 units is replaced by an -NH- unit, or

[0230] - at least one, preferably one, CH2 unit is replaced by an -SO2- unit, or

[0231] - at least two, preferably two, CH2 units are replaced by -O- and at least one, preferably one, CH2 unit is replaced by a -[SiR 2 R 3 O] m -SiR 2 R 3 - with R 2 , R 3 and m as described above.

[0232] More particularly, when one or more CH2 units are replaced by -O-, the replaced unit(s) are such that Y comprises one or more -CH2-CH2-O- units. In particular, Y comprises from 1 to 50 -CH2-CH2-O- units, advantageously from 2 to 25 -CH2-CH2-O- units, more advantageously from 2 to 15 -CH2-CH2-O- units. Y may comprise only -CH2-CH2-O- units.

[0233] More preferably, Y is an alkyl group comprising 1 to 150, in particular 1 to 50, in particular 1 to 20, for example 1 to 12, in particular 1 to 6 carbon atoms, preferably linear, in which optionally one or more CH2 units are replaced by one or more divalent units chosen from -O- and -NH-, more particularly between 1 and 50, in particular between 1 and 15, for example 1 or 2, divalent units chosen from -O- and -NH-.

[0234] According to a first embodiment, R 2 and the Rs 3, identical or different, represent a -OR group 11 with R 11 as described above. In particular, R 11 represents an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms, more particularly a (C1-C6)alkyl group.

[0235] According to a second embodiment, R 2 and the Rs 3 , identical or different, represent an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted (preferably unsubstituted) by one or more groups chosen from a halogen atom, an aryl or a hydroxyl, more preferably an unsubstituted (C1-C6)alkyl group such as a methyl or an ethyl.

[0236] Advantageously, the crosslinking agent is a compound of the following formula Chem. Ha: Z 1 -Y 1 -Z 2 in which the Z groups 1 and Z 2, identical or different, are chosen from isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide groups, and an acid anhydride residue, and Y 1 represents a divalent hydrocarbon chain, in particular aliphatic, comprising from 1 to 50 carbon atoms: - in which one or more (e.g. 1 to 15 or even 1 or 2) CH2 units are optionally replaced by one or more divalent units chosen from arylenes, -O-, -S-, -S(O)-, -C(=O)-, -SO2-, -N(R 1 )- , and -[SiR 2 R 3 O]m-SiR 2 R 3 - with

[0237] R 1 representing a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl, m an integer between 2 and 20; and

[0238] R 2 and the Rs 3 , identical or different, representing a hydrogen atom; a halogen atom; a -OR group 11 with R 11 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl,

[0239] - said chain being unsubstituted or substituted by one or more monovalent groups chosen from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl.

[0240] Z groups 1 and Z 2 have the same definition as group Z defined above.

[0241] In particular, the T group of the molecule of formula Chem. I and the Z groups 1 and Z 2of the molecule with the formula Chem. Ha are identical.

[0242] Y 1 has the same definition as Y defined above with valence n being equal to 2. In particular Y 1 may comprise only -CH2-CH2-O- units, as defined above.

[0243] Preferably, the crosslinking agent of formula Chem. II or Chem. Ha does not comprise -[SiR 2 R 3 O] m -SiR 2 R 3 -.

[0244] The crosslinking of step e) of the polysaccharide provided in step a) or of the polysaccharide obtained following step d) preferably takes place in the presence of 0.05 to 10 moles, in particular 0.05 to 7 moles, more advantageously 0.05 to 5 moles, even more advantageously 0.1 to 2 moles, of at least one crosslinking agent per 100 moles of repeating unit of the polysaccharide. In some embodiments, the crosslinking of step e) of the polysaccharide provided in step a) or of the polysaccharide obtained following step d) takes place in the presence of 0.1 to less than 2 moles, or 0.1 to 1.5 moles or 0.1 to 1 mole or 0.1 to 0.8 moles or 0.1 to 0.5 moles of at least one crosslinking agent per 100 moles of repeating unit of the polysaccharide.

[0245] In particular, the crosslinking of step e) takes place in an aqueous reaction medium. However, if necessary, an organic solvent such as an alcohol, in particular ethanol, or DMSO may be used to solubilize the crosslinking agent, for example when it is poly(dimethylsiloxane) terminated at each end by a diglycidyl ether (CAS number: 130167-23-6) before addition to the aqueous reaction medium.

[0246] Advantageously, and in particular when the groups Z, such as Z 1 or Z 2 , represent an epoxide group or a vinyl group, the crosslinking of step e) takes place at a pH greater than or equal to 10, more advantageously greater than or equal to 12.

[0247] For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide. In particular, the reaction medium comprises a Bronsted base, more preferably a hydroxide, even more preferably a sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0248] According to one embodiment, the crosslinking of step e) takes place between 4°C and 60°C, more preferably between 10°C and 50°C.

[0249] In particular, the crosslinking of step e) takes place between 1 hour and 2 weeks, more particularly between 3 hours and 1 week.

[0250] According to a variant, the crosslinking of step e) takes place at temperature T and pressure P, for a duration t as described above, the duration t varying according to the pH of the reaction medium. In this case, step e) is advantageously concomitant at least in part or totally with step d) and optionally step c).

[0251] In the presence of several crosslinking agents, the crosslinking agents may be added simultaneously or separately over time. Step e) may thus comprise repeated crosslinking steps. The total amount of crosslinking agents varies from 0.05 to 10 moles, in particular from 0.05 to 7 moles, more advantageously from 0.05 to 5 moles, even more advantageously from 0.1 to 2 moles, per 100 moles of repeating unit of the polysaccharide. In certain embodiments, the total amount of crosslinking agents varies from 0.1 to less than 2 moles, or from 0.1 to 1.5 moles or from 0.1 to 1 mole or from 0.1 to 0.8 moles or from 0.1 to 0.5 moles of crosslinking agents per 100 moles of repeating unit of the polysaccharide.

[0252] This step allows the polysaccharide chains to be crosslinked together. The functional groups of the crosslinking agent react with functional groups present on the polysaccharides so as to link the polysaccharide chains together and crosslink them by forming intermolecular bonds. The crosslinking agent can also react with functional groups present on the same polysaccharide molecule so as to form intramolecular bonds. In particular, the functional groups of the crosslinking agent react with the -OH or -COOH groups, optionally CHO, present on polysaccharides such as hyaluronic acid. Crosslinked polysaccharides are thus obtained comprising at least one crosslinking link between two polysaccharide chains, said crosslinking link being the residue of the crosslinking agent from step e).

[0253] In particular, following step e), the crosslinked polysaccharides comprise at least one crosslinking link between two polysaccharide chains, said crosslinking link more particularly comprising the polyvalent group Y as described above, preferably the divalent group Y 1 as described above.

[0254] Some functional groups Z (such as Z 1 and Z 2 ) of the crosslinking agent may however not react with a polysaccharide chain.

[0255] In particular, when the crosslinking agent comprises two functional groups Z 1 and Z 2 , one of the Z functional groups 1 can react with a polysaccharide while the other functional group Z 2 does not react with any polysaccharide. A dangling bond is then formed.

[0256] Generally speaking, the amount of functionalizing agent increases when the amount of crosslinking agent is decreased, and vice versa.

[0257] According to a first variant, step e) takes place before step c), in particular between step a) and step c).

[0258] According to a second variant, steps c) and e) are concomitant. According to this variant, step d) may be at least partly concomitant with step c) as described above.

[0259] According to a third variant, step e) takes place after step c) and before d) or after step c) and at least partly concomitantly with step d).

[0260] Thus, during step e), the reaction medium can be maintained in part preferably at temperature T and pressure P for a duration t as described above, this duration t varying according to the pH of the reaction medium, in particular when step e) is at least in part concomitant with step d).

[0261] According to a fourth variant, step e) takes place after step d).

[0262] In some embodiments, step e) is not implemented. Step f)

[0263] Preferably, the method according to the invention further comprises a step f) of adding a molecule of the following formula Chem. Ill:

[0264] R 7 GOLD 12 R 13 SiO] p -R 8 or a salt thereof in which:

[0265] - p is an integer from 1 to 20;

[0266] - R 12 and the Rs 13, identical or different, represent a hydrogen atom; a halogen atom; a -OR group 14 with R 14 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl; and

[0267] - R 7 and R 8 , identical or different, represent a hydrogen atom, an aryl group or an aliphatic hydrocarbon group containing from 1 to 6 carbon atoms.

[0268] According to one embodiment, step f) is carried out before the temperature of the reaction medium is at temperature T and pressure P during step d), in particular before, during or after step c).

[0269] According to a variant, step f) is carried out after step d), i.e. after the reaction medium has been maintained at temperature T and pressure P.

[0270] When step c) and step d) are concomitant, step f) is carried out before these steps c) and d), in particular between step a) and step c).

[0271] Preferably, R 12 and the Rs 13 , identical or different, represent a -OR group 14 with R 14 representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms optionally substituted by one or more groups chosen from a halogen atom, an aryl or a hydroxyl.

[0272] In particular, R 12 and the Rs 13 , identical or different, represent a -OR group 14 with R 14representing a hydrogen atom, an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably a (C1-C6)alkyl group; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably a (C1-C6)alkyl group.

[0273] Advantageously, R 7 and R 8 , identical or different, represent a hydrogen atom, or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms, preferably a (C1-C6)alkyl group.

[0274] This molecule with the formula Chem. Ill contains Si-OR groups (Si-OR 7 , Si-OR 8 and possibly Si-OR 14 ) capable of reacting with Si-OR groups (Si-OR 10 and possibly Si-OR 4) of the molecule of formula Chem. I. Thus, during the sol-gel reaction allowing the formation of Si-O-Si bonds, a molecule of formula Chem. Ill can bind to two molecules of formula Chem. I grafted onto polysaccharide chains so as to form crosslinking bonds resulting from the coupling of a molecule of formula Chem. Ill with two molecules of formula Chem. I.

[0275] For example, the molecule with the formula Chem. Ill is orthosilicic acid, tetraethyl orthosilicate (TEOS), polydimethylsiloxane (PDMS), oligomerized TEOS / orthosilicic acid, or methyl silanetriol (preferably used in the form of its sodium salt called sodium methyl siliconate - NAMS).

[0276] Advantageously, step f) takes place at a pH greater than or equal to 9, in particular greater than or equal to 10, more advantageously greater than or equal to 12, and in particular less than 14, for example less than or equal to 13.5, in particular when the molecule of formula Chem. Ill is sodium methyl siliconate (NAMS).

[0277] For this, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, even more preferably a sodium or potassium hydroxide. In particular, the reaction medium comprises a Bronsted base, more preferably a hydroxide, even more preferably a sodium or potassium hydroxide at a concentration of between 0.10M and 0.30M.

[0278] The following steps g) to i) are gel formulation steps carried out:

[0279] - either all before the reaction medium is at temperature T and pressure P during step d),

[0280] - either all after step d), i.e. after the reaction medium has been maintained at temperature T and pressure P. Step g)

[0281] Advantageously, the method according to the invention further comprises a step g) of adding at least one additional component chosen from lubricating agents; cosmetic active ingredients such as antioxidants, co-enzymes, amino acids, vitamins, minerals, and nucleic acids; and mixtures thereof, as described below.

[0282] Step g) may also comprise the addition of at least one therapeutic active ingredient advantageously chosen from anesthetics, antibiotics, antifungals, adrenaline and its derivatives, and mixtures thereof, as described below, in addition to or instead of the at least one additional component mentioned above.

[0283] Where appropriate, step g) preferably takes place after purification step h).

[0284] Where appropriate, step g) preferably takes place before step j) of sterilization.

[0285] In particular, step g) may also comprise the addition of at least one therapeutic active ingredient, at least one cosmetic active ingredient, or their mixture.

[0286] When at least one therapeutic active ingredient and / or at least one cosmetic active ingredient is added, step g) preferably takes place before the reaction medium is at temperature T and pressure P during step d).

[0287] Step h)

[0288] Preferably, the method further comprises at least one purification step h), in particular by dialysis.

[0289] Where applicable, at least one step h) preferably takes place before step g).

[0290] Where appropriate, at least one of step h) preferably takes place before step j) of sterilization.

[0291] Where appropriate, at least one of step h) preferably takes place before step i) of sieving.

[0292] According to a particular embodiment, a purification is carried out after step c). According to another embodiment, a purification is carried out after step e). According to another embodiment, a purification is carried out after step d).

[0293] Step i)

[0294] In particular, the method further comprises a step i) of sieving, more particularly with a sieve with a porosity of between 50 and 2000 μm. This sieving step makes it possible to obtain a more homogeneous hydrogel with the most constant extrusion force possible, i.e. the most regular possible. A person skilled in the art knows how to select a sieve with a suitable pore size depending on the mechanical properties of the gel that he wishes to obtain.

[0295] Preferably, step i) takes place after steps a) to d) and any steps e), f), g) and h).

[0296] Where appropriate, step i) preferably takes place before step j) of sterilization.

[0297] Step j)

[0298] The method according to the invention may comprise a sterilization step. Sterilization is preferably carried out by heat, in particular by autoclave. Sterilization is generally carried out by increasing the temperature of the sterilization medium to a temperature called "plateau temperature", which is maintained for a determined period of time called "plateau time". Sterilization is preferably carried out at a plateau temperature ranging from 121°C to 135°C, preferably with a plateau time ranging from 1 minute to 20 minutes with FO > 15. The sterilizing value FO corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the population of microorganisms present in the product to be sterilized. Alternatively, sterilization may be carried out in particular by gamma ray or UV radiation or by means of ethylene oxide.

[0299] Thus, advantageously, the method further comprises a step j) of sterilization of the hydrogel, in particular a step of sterilization by heat carried out at a temperature at the plateau of between 121°C and 135°C, preferably with a duration at the plateau of between 1 minute and 20 minutes with FO > 15 or a step of sterilization by UV radiation.

[0300] According to a first alternative, the sterilization step is carried out after steps a) to d) and any steps e), f), g), h) and i).

[0301] According to a second alternative, the sterilization step takes place after step c) and before the reaction medium is at temperature T and pressure P during step d). In this case, the pH of the reaction medium is adjusted to a physiological pH (6.8-7.8) before sterilization.

[0302] In particular, the gel is sterilized after being packaged in its injection device and the packaging of the gel takes place following all steps of the process and before sterilization. Step k)

[0303] In particular, the method further comprises a step k) of swelling the gel.

[0304] During the gel swelling step, the polysaccharide concentration of the gel is adjusted. In particular, a solvent is added, for example, water, phosphate buffer, water for injection. More specifically, the added solvent has a pH around physiological pH.

[0305] The polysaccharide concentration obtained following step k) is advantageously between 1 mg / g of gel and 50 mg / g of gel, more advantageously between 5 mg / g of gel and 35 mg / g of gel, even more advantageously between 10 mg / g of gel and 30 mg / g of gel.

[0306] Preferably, step k) takes place after step d) and the return of the reaction medium to a physiological pH (6.8-7.8), if necessary.

[0307] HYDROGEL

[0308] The present invention also relates to a hydrogel capable of being obtained by the method of the present invention. Such a hydrogel may also be designated by the term “cryogel”.

[0309] Preferably, the liquid medium of the hydrogel is an aqueous medium chosen from an aqueous solution or a mixture of aqueous solutions, preferentially chosen from water for injection, phosphate buffered saline or a mixture of the two, more preferentially phosphate buffered saline in the context of therapeutic, cosmetic and aesthetic applications according to the invention.

[0310] This hydrogel is preferably an injectable hydrogel. It is preferably sterile, in particular heat sterilized at a plateau temperature of between 121°C and 135°C, preferably with a plateau time of between 1 minute and 20 minutes with F0 > 15. The hydrogel is preferably homogeneous. The hydrogel is preferably stringy, with in particular a phase angle 5 of between 20° and 45°.

[0311] This hydrogel may also comprise an additional component selected from lubricating agents; cosmetic active ingredients such as antioxidants, co-enzymes, amino acids, vitamins, minerals, and nucleic acids; and mixtures thereof, as described below.

[0312] This hydrogel may also comprise at least one therapeutic active ingredient advantageously chosen from anesthetics, antibiotics, antifungals, adrenaline and its derivatives, and mixtures thereof, as described below. The polysaccharide of this hydrogel is preferably as defined above, within the framework of the description of step a) of the method according to the invention.

[0313] Preferably, a hydrogel according to the present invention, acceptable for the therapeutic and / or cosmetic applications targeted by the present invention, has a cross-over stress (or stress at the crossing of the moduli G' and G”) greater than or equal to 50 Pa, preferably between 50 and 5000 Pa and more preferably between 100 and 1000 Pa and an elastic modulus G' greater than or equal to 20 Pa, preferably from 100 Pa to 2000 Pa, more preferably from 100 Pa to 1000 Pa.

[0314] Preferably, a hydrogel according to the present invention, acceptable for the therapeutic and / or cosmetic applications targeted by the present invention, has a cohesiveness of 1 N to 30 N. This cohesiveness is measured by mechanical compression using a rheometer. For this, the gel is deposited on a Peltier plane with an initial air gap of 2.60 mm; it is then compressed at a constant speed of 100 pm / s up to 70% of the initial air gap, at 25°C; finally, the cohesiveness of the gel is measured at the end of the compression stroke. The more cohesive a gel is, i.e., the higher the cohesiveness value, the more it is able to withstand stresses, such as those it may encounter after its administration to a subject.

[0315] COMPOSITION

[0316] The present invention also relates to a composition comprising the hydrogel according to the present invention. It is preferably a cosmetic or pharmaceutical composition. It may further comprise physiologically acceptable excipients.

[0317] The hydrogel according to the invention comprises a polysaccharide, preferably hyaluronic acid, crosslinked. The composition may further comprise a polysaccharide, preferably hyaluronic acid, not crosslinked.

[0318] Non-crosslinked hyaluronic acid may be present in the composition as a lubricant.

[0319] The composition according to the present invention may thus comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight of polysaccharide, preferably hyaluronic acid, relative to the total weight of said composition, the polysaccharide such as hyaluronic acid, being present in crosslinked and optionally non-crosslinked form. In particular, the content of non-crosslinked polysaccharide, in particular hyaluronic acid, varies from 0 to 40% by weight, preferably from 1 to 40% by weight, more preferably from 5 to 30% by weight, relative to the total weight of polysaccharide, in particular hyaluronic acid, present in the composition.

[0320] The composition according to the present invention is preferably a sterile composition, in particular sterilized by heat at a plateau temperature of between 121°C and 135°C, preferably with a plateau time of between 1 minute and 20 minutes with FO > 15. It is preferably an injectable composition. The composition according to the invention then preferably comprises a physiologically acceptable medium, preferably a physiologically acceptable aqueous medium.

[0321] The physiologically acceptable aqueous medium may comprise a physiologically acceptable solvent or mixture of solvents and preferably comprises water, preferably the solvent is water.

[0322] The physiologically acceptable medium may also include isotonic agents such as oses, sodium chloride and their mixture.

[0323] The physiologically acceptable medium may further comprise at least one isotonic and physiologically acceptable saline solution.

[0324] Preferably, said balanced salt solution is a phosphate buffered saline solution, and particularly a KH2PO4 / K2HPO4 saline buffer.

[0325] The composition according to the invention may further comprise at least one additional compound chosen from lubricating agents; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; and mixtures thereof. Preferably, the additional compound is water-soluble or modified to be soluble in an aqueous medium. The composition according to the invention may also comprise at least one therapeutic active ingredient advantageously chosen from anesthetics, antibiotics, antifungals, adrenaline and its derivatives, and mixtures thereof, as described below. Preferably, the therapeutic active ingredient is water-soluble.

[0326] Examples of anesthetics include Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuvérine, Fomocaine, Guafécainol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, Proxymetacaine, Pyrrocaine, Quatacaine,Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine or a salt thereof, in particular a hydrochloride thereof, and a mixture thereof.,

[0327] Examples of antioxidants include, but are not limited to, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and their derivatives, and a mixture thereof.

[0328] Examples of amino acids include, but are not limited to, arginine (eg, L-arginine), isoleucine (eg, L-isoleucine), leucine (eg, L-leucine), lysine (eg, L-lysine or L-lysine monohydrate), glycine, valine (eg, L-valine), threonine (eg, L-threonine), proline (eg, L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (eg, N-acetylated derivatives such as N-acetyl-L-cysteine) and a mixture thereof.

[0329] Examples of vitamins and their salts include, but are not limited to, vitamins E, A, C, B, especially vitamins B6, B8, B4, B5, B9, B7, B12, and more preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0330] Examples of minerals include, but are not limited to, zinc salts (e.g., zinc acetate, in particular dehydrated), magnesium salts, calcium salts (e.g., hydroxyapatite, in particular in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g., copper sulfate, in particular pentahydrate), optionally in a hydrated form, and mixtures thereof.

[0331] As nucleic acids, mention may be made in particular of adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives and a mixture thereof.

[0332] Examples of coenzymes include coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

[0333] As adrenaline derivatives, noradrenaline can be mentioned.

[0334] The amounts of additional compounds depend of course on the nature of the compound in question, the desired effect, and the intended purpose of the composition as described herein. APPLICATIONS

[0335] The hydrogel or composition according to the invention may have therapeutic, cosmetic or aesthetic applications.

[0336] The present invention therefore also relates to a hydrogel or a composition according to the invention for its use in filling and / or replacing tissues, in particular soft tissues, in particular by injecting the hydrogel or the composition into the tissue.

[0337] The hydrogel or composition may be intended for superficial application. Superficial application refers to the administration of a composition into the upper layers of the skin, i.e., into or onto the skin, for example, by mesotherapy, for example, to reduce superficial wrinkles and / or to improve skin quality (such as skin radiance, density, or structure) and / or to rejuvenate the skin.

[0338] The hydrogel or composition may be intended for deep application. Deep application refers to the administration of a composition into the deeper layers of the skin and / or beneath the skin (above the periosteum) to increase soft tissue volume, such as to fill deep wrinkles and / or partially atrophied regions of the face and / or body.

[0339] The hydrogel or composition may be versatile, i.e., be used for both deep and superficial application.

[0340] Preferably, when the hydrogel or the composition according to the invention comprises at least one therapeutic active ingredient, the present invention relates to the hydrogel or a composition according to the invention for its use in the modified, delayed or prolonged release of therapeutic active ingredients.

[0341] In particular, the hydrogel or composition according to the invention is used in oral care and more particularly in the treatment of gingival recession, or to fill periodontal pockets. More particularly, the hydrogel or composition according to the invention is used to treat defects in gingival architecture that may occur with tooth loss, with aging, with periodontal diseases and disorders, or after the placement of tooth implants, crowns or bridges.The hydrogel or the composition according to the invention can also be used in ophthalmology, more particularly to protect ocular structures during eye surgery such as for example ophthalmic surgery of the anterior or posterior segment, cataract extraction possibly with implantation of an intraocular lens, corneal transplant surgery, glaucoma filtering surgery, or even implantation of a secondary lens. In this case, the hydrogel or the composition according to the invention will more particularly be injected into the eye.

[0342] The hydrogel or composition according to the invention can also be used in orthopedics or rheumatology, for example by injection into the synovial cavity. The hydrogel or composition according to the invention is then used as viscosupplementation.

[0343] The hydrogel or composition according to the invention can also be used in the treatment of lipodystrophy.

[0344] The hydrogel or composition according to the invention can be used in cosmetic surgery, in particular for gynecoplasties and / or penoplasties.

[0345] The hydrogel or composition according to the invention is administered more particularly by injection.

[0346] The hydrogel or composition according to the invention can also be used for the modified, delayed or prolonged release of therapeutic active ingredients, in particular the therapeutic active ingredients as described above. The longer the hydrogel is left at temperature T° and pressure P in the presence of the active ingredient(s), the greater the sol-gel reaction will be and the slower the release of the active ingredient will be. Thus, it is possible to adapt the duration and / or intensity of release of the active ingredient to the request. This also applies to the modified, delayed or prolonged release of cosmetic active ingredients.

[0347] The present invention also relates to the aesthetic, and therefore non-therapeutic, use of a hydrogel or a composition according to the invention to prevent and / or treat the alteration of the viscoelastic or biomechanical properties of the skin, and in particular to regenerate, moisturize, firm or restore the radiance of the skin, in particular by mesotherapy; to fill volume defects of the skin, and in particular to fill wrinkles, fine lines or scars (in particular hollow scars); to reduce the appearance of wrinkles and fine lines; or when said hydrogel or said composition comprises at least one cosmetic active ingredient, for the modified, delayed or prolonged release of cosmetic active ingredients, in particular as defined above.

[0348] For example, the present invention relates to the aesthetic use of a hydrogel or a composition according to the invention to reduce nasolabial folds and bitterness lines; to increase the volume of the cheekbones, chin or lips; to restore the volumes of the face, in particular the cheeks, temples, the oval of the face, and the area around the eyes; or to regenerate, hydrate, firm or restore the radiance of the skin, in particular by mesotherapy. In particular, the hydrogel or the composition according to the invention is an anti-aging hydrogel or composition. The hydrogel or the composition according to the invention is administered more particularly by injection.

[0349] The present invention also relates to a method for the cosmetic treatment, preferably anti-aging, of keratin materials, in particular of the skin, comprising at least one step of administering a hydrogel or a composition according to the invention on or through said keratin materials, more particularly by injection.

[0350] The administration may be an injection, in particular an intra-epidermal and / or intradermal and / or subcutaneous injection. The administration by intra-epidermal and / or intradermal and / or subcutaneous injection according to the invention aims to inject a hydrogel or a composition of the invention into an epidermal, dermo-epidermal and / or dermal region. The hydrogel or the composition according to the invention may also be administered by a supra-periosteal injection.

[0351] The hydrogel or composition according to the invention may be injected using any of the methods known to those skilled in the art. In particular, a hydrogel or composition according to the invention may be administered by means of an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supra-periosteal injection. The injection device may in particular be chosen from a syringe, a set of microsyringes, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller.

[0352] The injection device may comprise any commonly used injection means suitable for intraepidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. Preferably, such means may be a hypodermic needle or a cannula.

[0353] A needle or cannula according to the invention may have a diameter varying from 18 to 34 G, preferably between 25 and 32 G, and a length varying from 4 to 70 mm, and preferably from 4 to 25 mm. The needle or cannula is advantageously for single use.

[0354] Advantageously, the needle or cannula is associated with a syringe or any other device making it possible to deliver said hydrogel or said injectable composition through the needle or cannula.

[0355] According to an alternative embodiment, a catheter can be inserted between the needle / cannula and the syringe. In a known manner, the syringe can be operated manually by the practitioner or by a syringe holder such as guns.

[0356] Preferably, the injection device may be chosen from a syringe or a set of microsyringes. In an alternative embodiment, the injection device may be adapted to the mesotherapy technique.

[0357] Mesotherapy is a treatment technique involving intra-epidermal and / or intradermal and / or subcutaneous injection of a composition or hydrogel. The composition or hydrogel is administered using this technique by injection in the form of multiple small droplets at the level of the epidermis, the dermo-epidermal junction and / or the dermis in order, in particular, to create a subcutaneous coating. The mesotherapy technique is described in particular in the book "Traité de mésothérapie" by Jacques LE COZ, published by Masson, 2004. Mesotherapy performed on the face is also called mesolift, or also under the Anglo-Saxon term "mesoglow". Administration can also be topical.

[0358] Preferably, it is a topical application to the surface of the skin, more particularly to the epidermis, even more particularly to the facial epidermis.

[0359] The present invention thus also relates to an injection device as described previously comprising a hydrogel or a composition according to the invention.

[0360] The additional accessory biological effects of the hydrogels according to the invention can be studied in vitro and / or in vivo; said in vivo tests can for example include administration tests in small animals of a composition according to the invention vs. a comparative composition in order to monitor the appearance of biological effects with in particular the evaluation of the improvement in the quality of the skin in the animal, in particular living humans (e.g. its hydration and / or its elasticity) and, after sacrifice of the animal, histological sections to study the possible modification of protein expression at the administration site (coloration).

[0361] These in vivo tests may also include the evaluation of skin quality in humans following the administration of a composition according to the invention vs. a comparative composition.

[0362] Said in vitro tests include tests on dermal cells (such as fibroblasts) for cytotoxicity, viability, protein expression (ELISA) in particular for the expression of hyaluronic acid, elastin, fibrillin, aquaporin and / or collagens of different types and gene expression (eg, genes coding for hyaluronic acid, elastin, fibrillin, aquaporin and / or collagens of different types).

[0363] The present invention is illustrated by the non-limiting examples below. EXAMPLES

[0364] Materials and methods

[0365] - GPTMS: (3-Glycidyloxypropyl)trimethoxy-silane (Sigma 440167)

[0366] - APTES: (3-aminopropyl)triethoxysilane (SIGMA)

[0367] - NaHA: uncrosslinked sodium hyaluronate 1.5MDa and 4MDa (HTL)

[0368] - NaOH 0.25M

[0369] - 1M HCl (Chem Lab)

[0370] - PBS Phosphate Buffer (Braun),

[0371] - Lidocaine hydrochloride

[0372] - Turbula® three-dimensional agitator

[0373] - Homogenizer paddle mill

[0374] - Sterile bag

[0375] Measurement of viscoelastic properties

[0376] The viscoelastic properties of the obtained prototypes were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone-plate geometry and an anodized aluminum peltier plane (42 mm) (air gap 24 pm).

[0377] For the measurements of elastic modulus G', viscous modulus G” and phase angle 5, a stress sweep is carried out at 1 Hz and 25°C on the gel.

[0378] The elastic modulus G', the viscous modulus G” and the phase angle 5 are reported for a stress of 5 Pa.

[0379] The constraint at the intersection of G' and G” noted T hereafter is determined at the intersection of the curves of the modules G' and G”, and is expressed in Pascal.

[0380] Measurement of cohesiveness

[0381] To measure cohesiveness (or mechanical strength, expressed in Newtons), the gel is placed on the Peltier plane with an initial air gap of 2.60 mm. The gel is then compressed at a constant speed of 100 pm / s to 70% of the initial air gap, at 25°C. The cohesiveness of the gel is measured at the end of the compression stroke.

[0382] Extrusion force measurement

[0383] The extrusion forces (in Newton) of the syringe-packaged prototypes were conducted using a test bench (Mecmesin 2.5-dV) equipped with a dynamometer (Mecmesin AFG 100N) at a constant speed of 12.5 mm / min, over a distance of 2.5 cm, through a 27G 2” needle and at room temperature. The extrusion force results correspond to the average of the average extrusion forces on at least 3 samples.

[0384] Qualitative study of the stringy character of a product

[0385] 1 mL of the product under study is placed on a smooth, hard surface, e.g., a laboratory bench. Using their index finger, the operator exerts pressure on the product so as to compress it against the surface, then raises their index finger at a speed of about 1 cm / sec, to a height of about 2 cm from the surface. A photograph is taken when the index finger is at a height of about 2 cm from the surface. If the gel has broken, the gel is not stringy or has a weak stringy character. If the gel is not broken, it has an acceptable stringy character.

[0386] Unless otherwise stated, the steps described below are carried out at room temperature (21°C).

[0387] A month is 31 days.

[0388] Example 1: Study of the influence of the state, solid or liquid, of the reaction medium for functionalization and sol-gel reaction

[0389] Prototype No. 1 (comparative) and prototype No. 2 according to the invention are each prepared in the following manner:

[0390] 1- Dry sodium hyaluronate (NaHA) 4 MDa is dissolved at a concentration of 12% by weight in a 0.25M sodium hydroxide solution in a sterile bag,

[0391] 2- The mixture is homogenized,

[0392] 3- GPTMS is added to the sterile bag at a rate of 0.35 moles of GPTMS per 1 mole of NaHA repeat unit,

[0393] 4- The mixture is homogenized for 30 minutes,

[0394] 5- The bag containing the mixture is:

[0395] - for prototype 1: maintained at 21°C for 72 hours, the reaction medium is then in liquid form, the pH of the mixture is approximately 13,

[0396] - for prototype 2: placed in the freezer at -20°C for 2 months, the reaction medium is in the solid state, the pH of the mixture is approximately 13, then the bag is thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature, 6- A 1 N HCl solution is added to the sterile bag until a pH of 7 ± 0.5 is obtained,

[0397] 7- The mixture is diluted with water for injection to a concentration of 20 mg of hyaluronic acid / g of gel,

[0398] 8- The mixture is homogenized for 16 hours using a three-dimensional stirrer,

[0399] 9- The mixture is dialyzed,

[0400] 10- The gel thus obtained is packaged in a syringe,

[0401] 11- Finally, the gel is sterilized in an autoclave (FO > 15).

[0402] The viscoelastic properties of each prototype are analyzed. The results are presented in Table 1 below:

[0403] When the reaction medium of the concomitant functionalization and sol-gel reaction steps is liquid, at room temperature (prototype 1 - comparative), the prepared gel has lower viscoelastic properties (lower G' and stress at the intersection of G' and G”, increased phase angle 5) than when the medium is solid, frozen in accordance with the invention, at a temperature of -20°C (prototype 2 - in accordance with the invention).

[0404] Only the gel according to the invention (prototype 2) has acceptable properties for the therapeutic, cosmetic and aesthetic applications targeted by the present invention (stress at the intersection of G' and G” greater than 50 Pa (208 ± 25 Pa) and G' greater than 20 Pa (221 ± 21 Pa)). On the contrary, the comparative gel (prototype 1) does not have acceptable properties (stress at the intersection of G' and G” less than 50 Pa (29 ± 17 Pa)).

[0405] The state, liquid or solid, of the reaction medium during the concomitant steps of functionalization and sol-gel reaction therefore has an influence on the viscoelastic properties of the gels prepared. Example 2: Influence of temperature for the preparation of hydrogels according to the invention Prototypes No. 3 (according to the invention), No. 4 (comparative), No. 5 (comparative) are each prepared in the following manner:

[0406] 1- Dry sodium hyaluronate (NaHA) 1.5 MDa is dissolved at a concentration of 12% by weight in a 0.25M sodium hydroxide solution in a sterile bag,

[0407] 2- GPTMS is added to the sterile bag at a rate of 0.35 moles of GPTMS per 1 mole of NaHA repeat unit,

[0408] 3- The mixture is homogenized,

[0409] 4- The bag containing the mixture is:

[0410] - for prototype no. 3: placed in the freezer at -20°C for 1 month, the pH of the mixture is approximately 13, the reaction medium is then in the solid state, then the bag is thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature,

[0411] - for prototype no. 4: maintained at 21°C for 72 hours, the reaction medium is then in liquid form, the pH of the mixture is approximately 13,

[0412] - for prototype no. 5: maintained at 52°C for 3 hours, the reaction medium is then in liquid form, the pH of the mixture is approximately 13,

[0413] 5- A 1 N HCI solution is added to the sterile bag until a pH of 7 ± 0.5 is obtained,

[0414] 6- The mixture is diluted with phosphate buffered saline to a concentration of 23 mg of hyaluronic acid / g of gel,

[0415] 7- The mixture is homogenized for at least 16 hours and up to 24 hours using a three-dimensional agitator,

[0416] 8- The mixture is dialyzed,

[0417] 9- 1% by weight of a 30% by weight phosphate buffer solution of lidocaine hydrochloride and to counterbalance the acidic pH of lidocaine hydrochloride, and 0.4% by weight of a 0.25M NaOH solution are added to the dialyzed mixture,

[0418] 10- A fixed amount of uncrosslinked NaHA 4 MDa is added as a lubricant,

[0419] 11- The gel thus obtained is sieved,

[0420] 12- The gel thus obtained is packaged in a syringe,

[0421] 13- Finally, the gel is sterilized in an autoclave (F0 > 15). The viscoelastic properties of each prototype are analyzed. The results are presented in Table 2 below:

[0422] Table 2: Results

[0423] Only prototypes 3 and 4 are gels (phase angle less than 45°), injectable (extrusion forces between 5 and 25N) and have acceptable properties for therapeutic, cosmetic and aesthetic applications targeted by the present invention (stress at the intersection of G' and G” greater than 50 Pa and G' greater than 20 Pa).

[0424] However, for identical concentrations of reagents (GPTMS, NaHA), the prototype according to the invention (prototype 3) has higher viscoelastic properties and cohesiveness than the prototypes prepared according to the comparative methods (prototype 4 and 5).

[0425] In a similar manner to Example 1, when the reaction medium is frozen in accordance with the invention, at a temperature of -20°C (prototype 3), the prepared gel has a higher G' and stress at the intersection of G' and G” as well as a lower phase angle than a prototype prepared with a reaction medium that is liquid, at room temperature or at 52°C (prototypes 4 and 5 - comparative).

[0426] The state, liquid or solid, of the reaction medium during the concomitant stages of functionalization and sol-gel reaction therefore has an influence on the viscoelastic properties of the gels prepared. 3: Process according to the invention of based on hyaluronic acid and GPTMS comprising a crosslinking step with a crosslinking agent

[0427] Prototypes No. 6 (comparative) and No. 7 to 9 (according to the invention) are each prepared in the following manner:

[0428] 1- Dry sodium hyaluronate (NaHA) 1.5 MDa is dissolved at a concentration of 12% by weight in a 0.25M sodium hydroxide solution in a sterile bag,

[0429] 2- GPTMS is added to the sterile bag at a rate of 0.21 moles of GPTMS per 1 mole of NaHA repeating unit and 0.004 moles of BDDE per 1 mole of NaHA repeating unit,

[0430] 3- The mixture is homogenized for 30 minutes,

[0431] 4- The bag containing the mixture is:

[0432] - for prototype no. 6: maintained at 21°C for 72 hours, the reaction medium is then in liquid form, the pH of the mixture is approximately 13,

[0433] - for prototype no. 7: placed in the freezer at -20°C for 21 days, the pH of the mixture is approximately 13, the reaction medium is then in the solid state, then the bag is thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature,

[0434] - for prototype no. 8: placed in the freezer at -20°C for 30 days, the pH of the mixture is approximately 13, the reaction medium is then in the solid state, then the bag is thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature,

[0435] - for prototype no. 9: placed in the freezer at -20°C for 2 months, the pH of the mixture is approximately 13, the reaction medium is then in the solid state, then the bag is thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature,

[0436] 5- A 1 N HCI solution is added to the sterile bag until a pH of 7 ± 0.5 is obtained,

[0437] 6- The mixture is diluted with phosphate buffered saline to a concentration of 23 mg of HA / g of gel,

[0438] 7- The mixture is homogenized for at least 16 and up to 24 hours using a three-dimensional agitator, 8- The mixture is dialyzed,

[0439] 9- 1% by weight of a 30% by weight phosphate buffer saline solution in lidocaine hydrochloride and to counterbalance the acidic pH of lidocaine hydrochloride, 0.4% by weight of a 0.25M NaOH solution are added to the dialyzed mixture,

[0440] 10- a fixed amount of non-crosslinked NaHA 4 MDa is added as a lubricant,

[0441] 11- The gel thus obtained is sieved,

[0442] 12- The gel thus obtained is packaged in a syringe,

[0443] 13- Finally, the gel is sterilized in an autoclave (FO > 15).

[0444] The viscoelastic properties of each prototype are analyzed. The results are presented in Table 3 below. In addition, the stringiness of each prototype is studied qualitatively. The results obtained for prototypes 6 to 9 are presented in Figures 1 to 4, respectively.

[0445] Figure 1 shows the photo of the qualitative study of the stringing character of prototype 6.

[0446] Figure 2 shows the photo of the qualitative study of the running character of prototype 7.

[0447] Figure 3 shows the photo of the qualitative study of the running character of prototype 8.

[0448] Figure 4 shows the photo of the qualitative study of the running character of prototype 9.

[0449] Table 3: Results

[0450] All prototypes 6 to 9 are gels (phase angles less than 45°), injectable (extrusion forces between 5 and 25N) and have acceptable properties for therapeutic, cosmetic and aesthetic applications targeted by the present invention (stresses at the intersection of G' and G” greater than 50 Pa and G' greater than 20 Pa). Nevertheless, the process according to the invention results in the preparation of very stringy gels (prototypes 7 to 9), which is not the case for the comparative process. This stringy / non-stringy character is visible in the respective photographs of prototypes 6 to 9 in Figures 1 to 4.

[0451] Prototypes 7, 8 and 9 according to the invention have higher elastic properties (higher G' and lower phase angle) than comparative prototype 6.

[0452] The elastic properties of prototypes 7 to 9 increase progressively proportionally to the duration of the concomitant stages of functionalization, sol-gel reaction and crosslinking.

[0453] The state, liquid or solid, of the reaction medium during the concomitant stages of functionalization and sol-gel reaction therefore has an influence on the viscoelastic properties of the gels prepared.

[0454] Example 4: Methods for preparing gels based on hyaluronic acid and GPTMS using different levels of GPTMS functionalization

[0455] Prototypes No. 10 to 12 according to the invention are each prepared as follows:

[0456] 1- Dry sodium hyaluronate (NaHA) 1.5 MDa is dissolved at a concentration of 12% by weight in a 0.25M sodium hydroxide solution in a sterile bag,

[0457] 2- The GPTMS is added to the sterile bag respectively at the level of:

[0458] - for prototype no. 10: 0.1 moles of GPTMS for 1 mole of NaHA repeat unit,

[0459] - for prototype no. 11: 0.21 moles of GPTMS for 1 mole of NaHA repeat unit,

[0460] - for prototype no. 12: 0.35 moles of GPTMS for 1 mole of NaHA repeat unit,

[0461] 3- The mixture is homogenized,

[0462] 4- The bag containing the mixture is placed in the freezer at -20°C for 1 month except that of prototype 10 which is left for 3 months; the pH of the mixture is approximately 13, the reaction medium is then in the solid state, then the bag is thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature,

[0463] 5- A 1 N HCI solution is added to the sterile bag until a pH of 7 ± 0.5 is obtained, 6- The mixture is diluted with phosphate buffer saline to a concentration of 23 mg of HA / g of gel,

[0464] 7- The mixture is homogenized for at least 16 and up to 24 hours using a three-dimensional agitator,

[0465] 8- The mixture is dialyzed,

[0466] 9- 1% by weight of a 30% by weight lidocaine hydrochloride phosphate buffer solution and to counterbalance the acidic pH of lidocaine hydrochloride, and 0.4% by weight of a 0.25M NaOH solution are added to the dialyzed mixture;

[0467] 10- A fixed amount of uncrosslinked NaHA 4 MDa is added as a lubricant,

[0468] 11- The gel thus obtained is sieved,

[0469] 12- the gel obtained is packaged in a syringe,

[0470] 13- Finally, the gel is sterilized in an autoclave (FO > 15).

[0471] The viscoelastic properties of each prototype are analyzed. The results are presented in Table 4 below:

[0472] Table 4: Results

[0473] All prototypes 10 to 12 are gels (phase angles less than 45°), injectable (extrusion forces between 5 and 25N) and have acceptable properties for therapeutic, cosmetic and aesthetic applications targeted by the present invention (stresses at the intersection of G' and G” greater than 50 Pa and G' greater than 20 Pa).

[0474] In view of the above results, the elastic properties of the prototypes increase with the quantity of GPTMS used for the same freezing time (G' prototype 11, 21 mol% of GPTMS = 73 ± 7 Pa < G' prototype 12, 35 mol% of GPTMS = 176 ± 29 Pa). Example 5: Process for preparing a hydrogel based on hyaluronic acid and GPTMS in which only the advanced condensation step is carried out at -20°C Prototypes No. 13 and 14 according to the invention are each prepared as follows:

[0475] 1- Dry sodium hyaluronate (NaHA) 1.5 MDa is dissolved at a concentration of 12% by weight in a 0.25M sodium hydroxide solution in a sterile bag,

[0476] 2- GPTMS is added to the sterile bag respectively at a rate of 0.35 moles of GPTMS per 1 mole of NaHA repeat unit,

[0477] 3- The mixture is homogenized,

[0478] 4- The bag containing the mixture is placed at 21°C for 3 days, the pH of the mixture is approximately 13,

[0479] 5- Then,

[0480] - for prototype no. 13: the bag containing the mixture is placed at -20°C for 1 month, the pH of the mixture is approximately 13, the reaction medium is then in solid form; the mixture is then thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature,

[0481] - for prototype no. 14: step 6 follows step 4,

[0482] 6- A 1 N HCI solution is then added to the sterile bag until a pH of 7 ± 0.5 is obtained,

[0483] 7- The mixture is diluted with phosphate buffered saline to a concentration of 23 mg of HA / g of gel,

[0484] 8- The mixture is homogenized for at least 16 and up to 24 hours using a three-dimensional agitator,

[0485] 9- The mixture is dialyzed,

[0486] 10- 1% by weight of a 30% by weight lidocaine hydrochloride phosphate buffer solution and to counterbalance the acidic pH of lidocaine hydrochloride, 0.4% by weight of a 0.25M NaOH solution are added to the dialyzed mixture,

[0487] 11- A fixed amount of uncrosslinked NaHA 4 MDa is added as a lubricant,

[0488] 12- The gel thus obtained is sieved then packaged in a syringe,

[0489] 13- Finally, the gel is sterilized in an autoclave (F0 > 15).

[0490] The viscoelastic properties of each prototype are analyzed. The results are presented in Table 5 below:

[0491] Table 5: Results

[0492] The functionalization and freezing steps were separated in order to visualize their influence on the gel properties. We can thus observe that the freezing step improves the sol-gel reaction and thus obtains a gel with greater elastic properties.

[0493] Example 6: Influence of the molecular weight of the polysaccharide

[0494] Prototypes No. 15 to 18 according to the invention are each prepared as follows:

[0495] 1- Dry sodium hyaluronate (NaHA) 1.5 MDa for prototypes 15 and 16 or dry NaHA 4 MDa for prototypes 17 and 18 is dissolved at a concentration of 12% by weight in a 0.25M sodium hydroxide solution in a sterile bag,

[0496] 2- GPTMS is added to the sterile bag at a rate of 0.21 moles of GPTMS per 1 mole of NaHA repeat unit,

[0497] 3- The mixture is homogenized,

[0498] 4- The bag containing the mixture is placed in the freezer at -20°C for 1 month (for prototypes 15 and 17) or 2 months (for prototypes 16 and 18), the pH of the mixture is approximately 13, the reaction medium is then in the solid state,

[0499] 5- The mixture is thawed at room temperature by removing the bag from the freezer and leaving it at room temperature until the mixture in the bag returns to room temperature,

[0500] 6- A 1 N HCI solution is added to the sterile bag until a pH of 7 ± 0.5 is obtained,

[0501] 7- The mixture is diluted with phosphate buffered saline to a concentration of 23 mg of HA / g of gel,

[0502] 8- The mixture is homogenized for at least 16 and up to 24 hours using a three-dimensional agitator,

[0503] 9- The mixture is dialyzed,

[0504] 10- 1% by weight of a 30% by weight lidocaine hydrochloride phosphate buffer solution and to counterbalance the acidic pH of lidocaine hydrochloride, 0.4% by weight of a 0.25M NaOH solution are added to the dialyzed mixture,

[0505] 11- A given amount of uncrosslinked NaHA 4 MDa is added as a lubricant,

[0506] 12- The gel thus obtained is sieved then packaged in a syringe,

[0507] 13- Finally, the gel is sterilized in an autoclave (FO > 15).

[0508] The viscoelastic properties of each prototype are analyzed. The results are presented in Table 6 below and the study of the stringy character is visible in the respective photographs of prototypes 15 to 18 in Figures 5A, 5B, 6A, 6B, 7A, 7B, 8A and 8B.

[0509] Figure 5A shows the photo of 1 mL of prototype 15 deposited on a smooth, hard surface before the qualitative study of its stringy character.

[0510] Figure 5B shows the photo of the qualitative study of the stringy character of prototype 15. Figure 6A shows the photo of 1 mL of prototype 16 deposited on a smooth, hard surface before the qualitative study of its stringy character.

[0511] Figure 6B shows the photo of the qualitative study of the stringy character of prototype 16. Figure 7A shows the photo of 1 mL of prototype 17 deposited on a smooth, hard surface before the qualitative study of its stringy character.

[0512] Figure 7B shows the photo of the qualitative study of the stringy character of prototype 17. Figure 8A shows the photo of 1 mL of prototype 18 deposited on a smooth, hard surface before the qualitative study of its stringy character.

[0513] Figure 8B shows the photo of the qualitative study of the stringy character of prototype 18.

[0514] Table 6: Results

[0515] All prototypes 15 to 18 are gels (phase angles less than 45°). All prototypes 15 to 18 have acceptable properties for therapeutic, cosmetic and aesthetic applications targeted by the present invention (stresses at the intersection of G' and G” greater than 50 Pa and G' greater than 20 Pa).

[0516] The above results therefore show that the method according to the invention works using NaHAs of different molecular weights (1.5MDa and 4MDa).

[0517] It also emerges from the above results that by increasing the duration of the concomitant functionalization and sol-gel reaction steps according to the invention at -20°C from 1 to 2 months, the properties of the gels are reinforced, regardless of the molecular weight of the NaHA used initially.

[0518] Comparing prototypes 15 and 16 and 17 and 18 together, it appears that gels made with 4 MDa NaHA have both a larger G', a greater stress at the intersection of G' and G” (T) and a lower phase angle 5, i.e., a greater elastic part, than gels made with 1.5 MDa NaHA. It is therefore possible to obtain mechanically more resistant gels by using a higher molecular weight NaHA compared to a gel prepared according to the same method but using a lower molecular weight NaHA.

[0519] Therefore, only by using a 4 MDa NaHA instead of a 1.5 MDa NaHA, it is possible to decrease the amount of functionalizing agent required (here GPTMS) and / or to decrease the duration of the concomitant functionalization and sol-gel reaction steps to obtain a gel with identical mechanical properties in the end.

[0520] Example 7: Methods for preparing gels based on hyaluronic acid and APTES Prototypes No. 19 to 20 according to the invention are each prepared as follows:

[0521] 1- Dry sodium hyaluronate (NaHA) 1.5 MDa is dissolved at a concentration of 6.7% by weight in a 0.25M sodium hydroxide solution in a pot, 2- 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) are dissolved in 3.5 mL of water for injection to obtain, for each of these two reagents, a molar ratio in moles of reagent per mole of hyaluronic acid disaccharide unit of 0.5. The solution obtained is added to the hyaluronic acid solution. The mixture is stirred manually with a spatula for 5 minutes and then left to react for 30 min. The pH is then checked and, if necessary, adjusted to reach a pH equal to 6 with 1 M HCl or 0.25M NaOH,

[0522] 3- APTES is added to the mixture of hyaluronic acid, EDC and NHS dropwise to obtain a molar ratio of moles of APTES per mole of hyaluronic acid disaccharide unit of 0.2. The pH is monitored and, if necessary, adjusted to reach a pH between 4.5 and 6.5 with 1M HCl or 0.25M NaOH,

[0523] 4- The product is then placed in an oven for 15 hours at 21°C,

[0524] 5- At the end of these 15 hours the product is, if necessary, brought back to a pH between 6.8 and 7.8,

[0525] 6- Then, prototype 20 is placed for 48 hours at -20°C while prototype 19 is directly treated according to the following steps,

[0526] 7- The product is diluted with phosphate buffered saline to a concentration of 23 mg of HA / g of gel,

[0527] 8- The mixture is homogenized for at least 16 hours and up to 24 hours using a three-dimensional agitator,

[0528] 9- The mixture is dialyzed,

[0529] 10- A fixed amount of uncrosslinked NaHA 4 MDa is added as a lubricant,

[0530] 11- The gel thus obtained is sieved,

[0531] 12- The gel obtained is packaged in a syringe,

[0532] 13- Finally, the gel is sterilized in an autoclave (F0 > 15).

[0533] The viscoelastic properties of each prototype are analyzed. The results are presented in Table 7 below:

[0534] Table 7: Results While a condensation reaction by simply passing to a pH of 6.8 to 7.8 is not sufficient to prepare a gel with acceptable properties for applications targeted by the present invention (prototype 19 has a viscous predominance with a 5 greater than 45°, a modulus G” greater than the modulus G' including for low stresses and therefore no crossing of the curves of G' and G”), carrying out this condensation reaction in the frozen state (- 20°C, atmospheric pressure) and at a pH of 6.8 to 7.8 allows it (prototype 20 with stress at the crossing of G' and G” greater than 50 Pa and G' greater than 20 Pa).

Claims

DEMANDS 1. A process for preparing a hydrogel, preferably injectable, comprising the following steps: a) supplying at least one polysaccharide; b) supplying at least one molecule of Chem. I formula: or a salt thereof, in which: T represents an isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, or acid anhydride residue group; A represents a chemical bond or a spacer group; R 5 and R 6 These, whether identical or different, represent a hydrogen atom; a halogen atom; an -OR group 4 with R 4representing a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising from 1 to 6 carbon atoms possibly substituted by one or more group(s) selected from a halogen atom, an aryl and a hydroxyl; R 10 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms; c) functionalization of the polysaccharide with at least one molecule of Chem formula. I; d) sol-gel crosslinking of the functionalized polysaccharide to give a hydrogel; wherein step d) comprises sol-gel crosslinking carried out in a reaction medium at a pH greater than or equal to 9 and less than 14, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium such that measured at pressure P, for a period t ranging from 2 weeks to 17 weeks, or wherein step d) includes sol-gel reaction crosslinking carried out in a reaction medium at a pH greater than or equal to 6.8 and less than or equal to 7.8, at a pressure P less than or equal to atmospheric pressure and at a temperature T greater than the eutectic point temperature of the reaction medium as measured at pressure P and less than the freezing point temperature of the reaction medium as measured at pressure P, for a period t ranging from 1 h to 48 hours.

2. Method according to claim 1 wherein T is between -55°C and -5°C, preferably between -35°C and -10°C.

3. A process according to claim 1 or 2 wherein the polysaccharide is selected from pectin and pectic substances; chitosan; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan or chondroitin sulfate; and mixtures thereof.

4. A method according to any one of the preceding claims wherein the polysaccharide is a glycosaminoglycan, preferably hyaluronic acid or a salt thereof.

5. A method according to any one of the preceding claims, wherein the molecule of formula Chem. I is such that A is an aliphatic hydrocarbon divalent chain comprising from 1 to 12 carbon atoms, in which one or more divalent motifs selected from the arylenes -O-, -S-, -S(O)-, -C(=O)-, -SO2- and -N(R)- are optionally intercalated between two carbon atoms of said chain. 9 )- with R 9representing a hydrogen atom, an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl, said chain being unsubstituted or substituted by one or more monovalent groups selected from a halogen atom, a hydroxyl, an aryl-(C1-C6)alkyl.

6. A method according to any one of the preceding claims, wherein the molecule of formula Chem. I is such that: - T is as defined in claim 1 and advantageously represents a group amino or epoxide, preferably represents an epoxide group; - A is a divalent chain -(C1-C6)alkylene-O-(C1-C6)alkylene- such that -CH2-O- (CH2)3-; - R 5 and R 6 , identical or different, are each a grouping -OR 4 with R 4representing a (C1-C6)alkyl group, preferably a methyl or an ethyl group; or a (C1-C6)alkyl group, preferably a methyl or an ethyl group and - R 10 is a (C1-C6)alkyl group, preferably methyl or ethyl; the R groups 5 , R 6 and GOLD 10 preferably being identical.

7. A process according to any one of the preceding claims, wherein in step c), said polysaccharide is functionalized in the presence of 0.01 to 0.5, preferably 0.03 to 0.3, again preferably 0.04 to 0.18 moles of a molecule of formula Chem. I or a salt thereof for 1 mole of repeating unit of the polysaccharide.

8. A method according to any one of the preceding claims, wherein steps c) and d) are concurrent or partly concurrent.

9. A process according to any one of the preceding claims, further comprising a step e) of crosslinking the polysaccharide in the presence of at least one crosslinking agent or a salt thereof, said crosslinking agent comprising at least two identical or different Z functional groups selected from the following groups: isocyanate, amino, epoxide, carboxyl, N-succinimidyloxycarbonyl, N-sulfosuccinimidyloxycarbonyl, halocarbonyl, isothiocyanate, vinyl, formyl, hydroxyl, sulfhydryl, hydrazino, acylhydrazino, aminoxy, carbodiimide, and an acid anhydride residue.

10. A method according to any one of the preceding claims, further comprising a step (f) of adding a molecule of Chem. III formula: R 7 GOLD 12 R 13 SiO] p -R 8 or a salt thereof in which: - p is an integer from 1 to 20; - R 12 and R 13These, whether identical or different, represent a hydrogen atom; a halogen atom; an -OR group 14 with R 14 representing a hydrogen atom, a an aryl group or an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms; an aryl; or an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms optionally substituted by one or more groups selected from a halogen atom, an aryl, or a hydroxyl; and - R 7 and R 8 , identical or different, represent a hydrogen atom, an aryl group or an aliphatic hydrocarbon group comprising 1 to 6 carbon atoms.

11. A method according to any one of the preceding claims, further comprising a step j) of sterilizing the hydrogel.

12. A method according to any one of the preceding claims, further comprising a step (g) of adding at least one additional component selected from lubricating agents; cosmetic active ingredients such as antioxidants, coenzymes, amino acids, vitamins, minerals, and nucleic acids; and mixtures thereof.

13. A method according to any one of the preceding claims, also comprising the addition of at least one therapeutic active ingredient, advantageously selected from anesthetics, antibiotics, antifungals, adrenaline and its derivatives, and mixtures thereof.

14. Hydrogel obtainable by a process according to any one of claims 1 to 13.

15. Cosmetic or pharmaceutical composition comprising a hydrogel according to claim 14.

16. Hydrogel according to claim 14 or composition according to claim 15 for its use in filling and / or replacing tissues.

17. Aesthetic use of a hydrogel according to claim 14 or a composition according to claim 15 to prevent and / or treat alterations in the viscoelastic or biomechanical properties of the skin; to fill volumetric defects of the skin, in particular to fill wrinkles, fine lines and scars; to reduce nasolabial folds and marionette lines; to increase the volume of cheekbones, chin or lips; to restore facial volume, particularly of the cheeks, temples, oval of the face, and around the eyes; to reduce the appearance of wrinkles and fine lines; or to regenerate, hydrate, firm or restore the radiance of the skin, particularly by mesotherapy.

18. Aesthetic use of a hydrogel according to claim 14 or of a composition according to claim 15, said hydrogel or said composition comprising at least one cosmetic active ingredient, for the modified, delayed or prolonged release of cosmetic active ingredients.

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