A process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof.

Abstract

The present invention relates to a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof, in particular comprising cross-linked hyaluronic acid, non-cross-linked hyaluronic acid, or a mixture thereof, and further comprising zinc and citrate ions, as well as a method for its preparation. Abstract figure: Figure 1

Description

Title of the invention: Process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof. FIELD OF THE INVENTION

[0001] The present invention relates to a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, in particular comprising a cross-linked hyaluronic acid, a non-cross-linked hyaluronic acid or a mixture thereof, and further comprising zinc and citrate ions, as well as a method for its preparation. TECHNOLOGICAL BACKGROUND

[0002] Polysaccharides, such as glycosaminoglycans, are widely used in the medical and aesthetic fields, particularly for filling soft tissues. In particular, the majority of products marketed for aesthetic applications are hyaluronic acid-based. To improve skin quality, hydrogels prepared from unmodified hyaluronic acid are of interest because they have the advantage of being perfectly biocompatible.

[0003] It is also possible to use hydrogels based on modified hyaluronic acid, the hyaluronic acid usually being modified by cross-linking. This cross-linking has the advantage of increasing the in vivo durability and resistance to in vivo degradation of the hydrogels. Cross-linked hyaluronic acid-based hydrogels can be obtained by various preparation processes.

[0004] Today, there is a growing need for hydrogels capable of improving their biocompatibility profile and delivering beneficial biological effects for skin quality. In this context, zinc is proving to be an ideal element. It is a micronutrient with numerous beneficial biological effects as a cofactor for many enzymes, particularly those involved in wound healing and extracellular matrix reconstruction. In addition to its healing activities, anti-inflammatory and anti-infective properties have also been associated with zinc. Zinc may therefore be of interest in reducing potential side effects due to the inflammatory response associated with the administration of hydrogels.

[0005] However, the preparation of hydrogels containing zinc is not easy, as zinc can precipitate in the presence of certain salts, particularly phosphates, carbonates, and / or sulfates. Its incorporation into hydrogels therefore remains difficult.

[0006] Thus, a need remains for the provision of a process enabling To prepare hydrogels comprising a cross-linked and / or non-cross-linked polysaccharide and further comprising zinc, particularly at concentrations where the zinc is biologically active, without precipitation of the latter. Advantageously, the proposed process will be as respectful as possible of the properties of the hydrogels, that is to say, it will cause the least possible degradation of the rheological properties of the hydrogels during heat sterilization. BRIEF DESCRIPTION OF THE INVENTION

[0007] The present invention relates to a process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof and further comprising zinc ions, the process comprising the following steps:

[0008] (1) preparation of a hydrogel comprising a cross-linked polysaccharide, a poly non-crosslinked saccharide or their mixture, the preparation of the hydrogel comprising the following steps:

[0009] - bringing the cross-linked polysaccharide into contact with the non-cross-linked polysaccharide, or with their mixture, with a physiological saline solution, preferably buffered, the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof,

[0010] - addition of citrate ions to the cross-linked polysaccharide, to the non-cross-linked polysaccharide, or to a mixture thereof, in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel,

[0011] - addition of zinc ions to the cross-linked polysaccharide, to the non-cross-linked polysaccharide, or to their mixture, in sufficient quantity to achieve a zinc ion concentration of no more than 20 mM in the hydrogel,

[0012] the addition of citrate ions and zinc ions being carried out in a molar ratio [citrate ions] / [zinc ions] ranging from 1 to 20, and

[0013] provided that the addition of zinc ions is not carried out before the addition of citrate ions when the contact with the physiological saline solution, preferably buffered, is carried out before the addition of citrate ions;

[0014] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of the step (1) to obtain a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof and further comprising zinc ions.

[0015] The invention also relates to a method for preparing a sterile hydrogel comprising a cross-linked polysaccharide and optionally a non-cross-linked polysaccharide and further comprising zinc ions, the method comprising the following steps:

[0016] (0) preparation of a crosslinked polysaccharide from a reticulated reaction medium culation comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and zinc ions in an amount allowing the preparation of a hydrogel comprising at most 20 mM of zinc ions;

[0017] (1) preparation of a hydrogel from the cross-linked polysaccharide obtained at the end of step (0) and possibly a non-crosslinked polysaccharide, the preparation of the hydrogel comprising a step of contacting the crosslinked polysaccharide with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0018] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of the step (1) to obtain a sterile hydrogel;

[0019] in which:

[0020] - the crosslinking reaction medium further comprises citrate ions in a sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, with a molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0021] - step (1) further comprises, before the step of contacting the polysaccharide crosslinked with physiological saline solution, preferably buffered, a step of adding citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions added] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0022] - the physiological saline solution, preferably buffered, further comprises citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] ranging from 1 to 20.

[0023] The invention also relates to a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, in particular comprising a cross-linked hyaluronic acid, a non-cross-linked hyaluronic acid or a mixture thereof, and further comprising zinc and citrate ions obtained by the processes according to the invention.

[0024] The invention relates to the use of citrate ions to protect a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, optionally an anesthetic agent, and furthermore zinc ions, from the degradation of its rheological properties during its sterilization, preferably by heat or to preserve the stability over time of the rheological properties of a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, optionally an anesthetic agent, and furthermore zinc ions.

[0025] Finally, the invention relates to the use of a solution comprising zinc ions and citrate ions to protect a hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide, optionally an anesthetic agent, from the degradation of its rheological properties during sterilization, preferably by heat, or to preserve the stability over time of a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof, possibly an anesthetic agent.

[0026] Other aspects of the invention are as described below and in the claims. FIGURES

[0027] [Fig.1] hydrogel obtained according to the process of the invention (microscope: Olympus SZX16, software: OLYMPUS Stream Start)

[0028] [Fig.2] hydrogel outside the invention (microscope:: Olympus SZX16, software: OLYMPUS Stream Start) DETAILED DESCRIPTION OF THE INVENTION Definitions

[0029] The term "gel" refers to a network of polymers 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 within each other. The so-called "solid" medium consists of long polymer molecules connected to each other by weak bonds (for example, hydrogen bonds) or by covalent bonds (cross-linking). The liquid medium consists of a solvent. A gel generally corresponds to a viscoelastic product that has a phase angle θ of less than 90°, preferably less than or equal to 70°, preferably less than or equal to 45°, at 1 Hz for a strain of 0.1% or a pressure of 1 Pa, preferably a phase angle θ ranging from 2° to 45° or from 20° to 45°.

[0030] The term “hydrogel” refers to 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%, especially at least 97%, especially at least 98% by weight of the liquid medium) and having a pH ranging from 6.8 to 7.8.

[0031] The term “injectable hydrogel” refers to a hydrogel that can flow and be manually injected using a syringe fitted with a needle with a diameter of 0.1 to 0.5 mm, for example a 32G, 30G, 27G, 26G, 25G hypodermic needle. Preferably, an “injectable hydrogel” is a hydrogel having an average extrusion force less than or equal to 25N, preferably from 5 to 25N, more preferably from 8 to 15N, when measured with a dynamometer, at a fixed speed of about 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 (27G) and of length U2”, at room temperature.

[0032] A “superficial application” refers to administration, for example by meso therapy, of a composition superficially in the skin, or on the skin, for the treatment of the superficial layers of the skin, the epidermis and the most superficial parts of the dermis, to reduce superficial wrinkles and / or improve skin quality (such as its radiance, density or structure) and / or rejuvenate the skin.

[0033] A “midline application” refers to the administration of a composition into the midline of the skin to treat the midline layers of the skin, as well as to reduce midline wrinkles.

[0034] A “deep application” refers to the administration of a hydrogel into the deepest layers of the skin, the hypodermis and the deepest part of the dermis, and / or under the skin (above the periosteum) to “add volume,” such as for filling the deepest wrinkles and / or partially atrophied areas of the facial and / or body contour. So-called “volumizing” hydrogels can typically be administered for deep application.

[0035] A “crosslinked polysaccharide” refers to a polysaccharide modified during a crosslinking reaction.

[0036] Conversely, a "non-crosslinked polysaccharide" refers to a polysaccharide that has not been modified with a crosslinking agent and therefore has not undergone a crosslinking reaction.

[0037] The term “crosslinking agent” refers to any compound capable of introducing crosslinking between different polysaccharide chains.

[0038] The "molar crosslinking ratio" (CR), expressed as a percentage, refers to the molar ratio of the amount of crosslinking agent to the amount of polysaccharide repeat units introduced into the crosslinking reaction medium, expressed per 100 moles of polysaccharide repeat units in the crosslinking medium. For example, a molar crosslinking ratio of 1% means that there is one molecule of crosslinking agent introduced into the reaction medium for every 100 polysaccharide repeat units.

[0039] The expression "repeat unit" of a polysaccharide refers to a structural motif consisting of one or more (usually 1 or 2) monosaccharides whose repetition produces the complete polysaccharide chain.

[0040] The "degree of modification" (MOD) of a polysaccharide, such as hyaluronic acid, corresponds to the molar amount of crosslinking agent bound to the polysaccharide, at one or more of its ends, expressed per 100 moles of polysaccharide repeat units. It can be determined by methods known to those skilled in the art, such as Nuclear Magnetic Resonance (NMR) spectroscopy. For example, a degree of modification of 1% means that there is one molecule of crosslinking agent per 100 polysaccharide repeat units.

[0041] The term “polysaccharide” refers to a polymer composed of monosaccharides (preferably D enantiomers) joined together by glycosidic bonds.

[0042] By "ambient temperature", it is understood that a temperature ranging from 20 to 25°C, more specifically 21°C.

[0043] The linear viscoelastic region (LVER) corresponds to the range of hydrogel deformations from an initial elastic modulus value G' to a value of the elastic modulus G' reduced by 10% of its initial value. The LVER measurement consists of an oscillatory stress scan measurement in compression mode at a given oscillation frequency to determine the linear viscoelastic region. Processes

[0044] The inventors have developed two alternative processes that meet the expressed needs.

[0045] According to a first variant (method 1), the preparation of a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide and further comprising zinc is made possible by the addition of citrate ions during the hydrogel preparation phase.

[0046] According to a second variant (method 2), when the hydrogel comprises a crosslinked polysaccharide, the preparation of a hydrogel comprising zinc is made possible by conducting the crosslinking of the polysaccharide in a reaction medium comprising zinc ions and by adding citrate ions either during the crosslinking step or subsequently but before any contact of the zinc ions with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts, alone or mixtures thereof.

[0047] It has been observed that the addition of citrate ions, in particular citric acid, to zinc prevents the precipitation of zinc, especially in the presence of a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof.

[0048] Unexpectedly, it was observed that the proposed process (variants 1 and 2) also effectively protects the hydrogel from degradation of its rheological properties during sterilization, particularly during heat sterilization. Hydrogels obtained by the process of the present invention thus exhibit less modification of their rheological properties after sterilization (better preservation of the elastic modulus G', better preservation of the phase angle) compared to hydrogels prepared by an equivalent process without the addition of zinc and citrate ions, in particular without the addition of zinc and citrate ions in the form of a physiological saline solution, preferably buffered, comprising phosphate, carbonate, or sulfate salts or mixtures thereof.

[0049] Unexpectedly, this protective effect is long-lasting. The hydrogels obtained by the process of the present invention exhibit better stability over time, that is to say, they maintain their rheological properties more effectively over time, particularly after sterilization, than hydrogels prepared by an equivalent process without the addition of zinc and citrate ions, in particular without the addition of zinc and citrate ions in the form of a physiological saline solution, preferably buffered, comprising phosphate, carbonate, or sulfate salts or mixtures thereof, and zinc and citrate ions. Indeed, the hydrogels according to the invention exhibit better long-term retention of their rheological properties, particularly after sterilization, and do not exhibit zinc precipitation.

[0050] Furthermore, it is known that the additional presence of an anesthetic in a hydrogel during sterilization, preferably by heat, leads to degradation of the rheological properties of hydrogels based on crosslinked and / or non-crosslinked polysaccharides, in particular hydrogels based on crosslinked and / or non-crosslinked hyaluronic acid. The addition of citrate and zinc ions helps to limit these effects. Hydrogels obtained by the process of the present invention comprising an anesthetic agent exhibit less degradation of their rheological properties after sterilization compared to hydrogels, comprising an anesthetic agent, prepared by an equivalent process without the addition of zinc and citrate ions, in particular without the addition of zinc and citrate ions in the form of a physiological saline solution, preferably buffered, comprising phosphate, carbonate, or sulfate salts or mixtures thereof. Method 1

[0051] The present invention thus relates to a process for preparing a sterile hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide and further comprising zinc ions, the process comprising the following steps:

[0052] (1) preparation of a hydrogel comprising a cross-linked and / or non-cross-linked polysaccharide crosslinked, the preparation of the hydrogel includes the following steps:

[0053] - contacting a cross-linked and / or non-cross-linked polysaccharide with a solution physiological saline, preferably buffered, the saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0054] - additions of zinc ions and citrate ions to the cross-linked and / or non-cross-linked polysaccharide in a molar ratio of citrate ions / zinc ions ranging from 1 to 20 and in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel and a zinc ion concentration of at most 20 mM in the hydrogel,

[0055] provided that the addition of zinc ions is not carried out before the addition of citrate ions when contact with the saline physiological solution is carried out before the addition of citrate ions;

[0056] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of the step (1) to obtain a sterile hydrogel.

[0057] It is understood that the preparation of the hydrogel from the cross-linked polysaccharide and / or non-crosslinked (step (1)) includes at least the steps indicated above, the order of these steps being irrelevant. Other steps may be implemented. The crosslinked and / or non-crosslinked polysaccharide

[0058] The polysaccharide can be any polymer composed of monosaccharides joined together by glycosidic bonds or mixtures thereof. Preferably, the polysaccharide is selected from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosane, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof. Even more preferably, the polysaccharide is chosen from hyaluronic acid, heparosane, chondroitin and mixtures thereof, even more preferably the polysaccharide is hyaluronic acid or one of its salts, in particular a physiologically acceptable salt such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and mixtures thereof.More specifically, hyaluronic acid is available in its acidic form or as a sodium salt (NaHA). A hydrogel can therefore be a hydrogel based on hyaluronic acid and / or one of its salts.

[0059] Preferably, if the polysaccharide is hyaluronic acid or one of its salts, it has a weight average molecular mass (Mw) ranging from 0.05 to 10 MDa, preferably ranging from 0.5 to 5 MDa, for example ranging from 2 to 4 MDa or ranging from 1 to 5 MDa.

[0060] The polysaccharide can be supplied in hydrated form (fully or partially hydrated), or in dry form, such as powder or fiber. When the polysaccharide is supplied in hydrated form, it is typically in the form of a gel.

[0061] A cross-linked polysaccharide can be prepared by any method known to those skilled in the art. For example, the cross-linked polysaccharide can be prepared as described in WO2010131175A1 or WO201277054A1.

[0062] The process of the present invention may thus include, before the hydrogel preparation step, a step of preparing a cross-linked polysaccharide.

[0063] The cross-linked polysaccharide is preferably a cross-linked polysaccharide with a molar degree of cross-linking less than or equal to 10%. Preferably, the cross-linked polysaccharide is a cross-linked polysaccharide with a molar degree of cross-linking greater than 0 and less than or equal to 6%. Even more preferably, the cross-linked polysaccharide is a cross-linked polysaccharide with a molar degree of cross-linking greater than 0 and less than or equal to 4%. Even more preferably, the cross-linked polysaccharide is a cross-linked polysaccharide with a molar degree of cross-linking greater than 0 and less than or equal to 2%, preferably less than or equal to 1%. preferably less than or equal to 0.8%, in particular ranging from 0.1% to 0.5% (number of moles of crosslinking agent(s) per 100 moles of repeat unit of the polysaccharide(s)).

[0064] The polysaccharide can be crosslinked by reacting a previously modified polysaccharide. The polysaccharide may have been modified by grafting with a molecule that allows for subsequent crosslinking of the modified polysaccharide. For example, the polysaccharide may have been modified by grafting a silylated molecule, an amino acid, an amino acid derivative, or a protein.

[0065] The polysaccharide is preferably crosslinked using a crosslinking agent selected from bi- or multi-functional epoxy or non-epoxy crosslinking agents, i.e., prepared by reaction of the polysaccharide with a crosslinking agent. Among the epoxy agents, 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxyoctane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol)-diglycidyl ether (PEGDE), and mixtures thereof may be mentioned. Examples of non-epoxy agents include endogenous polyamines such as spermine, spermidine and putrescine, aldehydes such as glutaraldehyde, carbodiimides and divinylsulfone, hydrazide derivatives such as adipic acid dihydrazide, bisalkoxyamines, dithiols such as polyethylene glycol dithiol and mixtures thereof.Examples of non-epoxide agents include amino acids such as cysteine ​​and lysine; peptides or proteins containing amino acids such as cysteine ​​and lysine; poly(dimethylsiloxane); and trimetaphosphates, such as sodium trimetaphosphate, calcium trimetaphosphate, and barium trimetaphosphate.

[0066] In some embodiments, the crosslinking agent is an epoxy agent, preferably 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol diglycidyl ether. Preferably, the crosslinking agent is 1,4-butanediol diglycidyl ether (BDDE).

[0067] In some embodiments, the crosslinking agent is a non-epoxy agent, preferably selected from endogenous polyamines, aldehydes, carbodiimides, divinyl sulfone, amino acids, peptides and mixtures thereof.

[0068] The cross-linked polysaccharide is preferably a cross-linked polysaccharide having a degree of modification (MOD) of 10% or less, preferably 6% or less, preferably 4% or less, preferably 2% or less, and more preferably 1% or less. Advantageously, the cross-linked polysaccharide is a cross-linked polysaccharide having a degree of modification (MOD) of 1.8% or less, more preferably 1.5% or less, preferably 1.2% or less, and even more preferably less than 1%.

[0069] The cross-linked polysaccharide can in particular be prepared by a process comprising the following steps:

[0070] (al) prepare a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s) and a solvent; and

[0071] (a2) react the reaction medium to obtain a cross-linked polysaccharide.

[0072] The polysaccharide is as described above. Preferably, the polysaccharide is hyaluronic acid or a salt of hyaluronic acid, preferably a sodium salt.

[0073] In step (a1a), the polysaccharide may be supplied in dry form, such as powder or fibers. When the polysaccharide is supplied 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 specifically, the polysaccharide is mixed with water, optionally with the addition of an alkaline medium suitable for crosslinking or a phosphate buffer or a supplemented phosphate buffer, i.e., possibly comprising additional components as defined above.

[0074] The crosslinking agent is as described above.

[0075] The solvent is typically water or a mixture comprising water and an organic solvent (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). For example, 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 its addition to the aqueous reaction medium.

[0076] The reaction medium may further comprise salts, pH adjusters, for example a Brønsted base, more preferably a hydroxide salt, such as sodium or potassium hydroxide, additional components as described above, and mixtures thereof. The addition of a Brønsted base may be particularly necessary when the functional groups of the crosslinking agent have an epoxide or vinyl group. In these cases, crosslinking occurs at a pH greater than or equal to 10, more advantageously greater than or equal to 12, which requires the addition of a Brønsted base to the reaction medium, typically at a concentration between 0.10 M and 0.30 M.

[0077] The total amount of crosslinking agent in the reaction medium typically varies from 0.001 to 0.10 moles per 1 mole of polysaccharide repeat unit, preferably from 0.001 to 0.08 or from 0.001 to 0.06 moles per 1 mole of polysaccharide repeat unit, preferably from 0.001 to 0.04 moles per 1 mole of polysaccharide repeat unit, preferably from 0.001 to 0.03 moles per 1 mole of polysaccharide repeat unit, preferably from 0.001 to 0.02 moles per 1 mole of repeat unit of the polysaccharide, more preferably from 0.001 to 0.01 moles per 1 mole of repeating unit of the polysaccharide, and even more preferably from 0.001 to 0.005 moles per 1 mole of repeating unit of the polysaccharide. When the polysaccharide is a glycosaminoglycan such as hyaluronic acid, the repeating unit is a disaccharide unit.

[0078] The mass concentration of polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 80 to 200 mg / g.

[0079] Step (al) of the process typically includes a step of homogenizing the reaction medium. Homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with paddles or stirring with a spatula.

[0080] Step (a1a) is typically carried out at a temperature ranging from 4 to 35°C, preferably from 15°C to 25°C. Preferably, the duration of step (1) does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 minutes to 2 hours.

[0081] Step (a2) consists of reacting the reaction medium to obtain a cross-linked polysaccharide. Advantageously, step (a2) is carried out directly after step (a1).

[0082] This step cross-links the polysaccharide chains together. The functional groups of the cross-linking agent react with functional groups present on the polysaccharides to link the polysaccharide chains together and cross-link them by forming intermolecular bonds. The cross-linking agent can also react with functional groups present on the same polysaccharide molecule to form intramolecular bonds. In particular, the functional groups of the cross-linking agent react with the -OH, -COOH, or -CHO groups present on polysaccharides such as hyaluronic acid. Cross-linked polysaccharides comprising at least one cross-link between two polysaccharide chains, said cross-link being the residue of the cross-linking agent, are thus obtained.

[0083] Crosslinking can be carried out in the presence of several crosslinking agents. When crosslinking is carried out in the presence of several crosslinking agents, the crosslinking agents can be added simultaneously or separately in time to the reaction medium. Step (a2) can thus comprise repeated crosslinking steps; advantageously, step (a2) comprises a single crosslinking step. Crosslinking is then carried out in the presence of a total quantity of crosslinking agents typically ranging from 0.1 to 10 moles, or from 0.1 to 8 moles, or from 0.1 to 6 moles, or from 0.1 to 4 moles, or from 0.1 to 3 moles, or from 0.1 to 2 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 (or their salts) per 100 moles of unit of polysaccharide repetition. The crosslinking conditions, in particular the crosslinking agent content, duration and temperatures as well as the weight average molecular masses (Mw) of the polysaccharide, used are interdependent.

[0084] The lower the crosslinking agent content, the longer the reaction time must be to obtain similar mechanical properties of the resulting crosslinked polysaccharide, and ultimately of the prepared hydrogel. In other words, the lower the molar percentage of crosslinking agent, the fewer reactive functional groups there are in the reaction medium and the lower the probability that two groups will meet and react together. Thus, the longer the reaction time must be to allow the functional groups to react with each other and form crosslinking bonds, thereby obtaining a crosslinked polysaccharide, and ultimately a hydrogel with desirable properties.

[0085] In some embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1a) at a temperature of 30°C or lower, preferably 25°C or lower. The temperature is typically above 0°C, above 5°C, or above 10°C. Even more preferably, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1a) at room temperature. When step (a2) is carried out at a temperature of 30°C or lower but above 0°C, the crosslinking time is at least 1 minute, preferably at least 10 minutes, and even more preferably at least 1 hour. Preferably, the crosslinking time is at most 5 days.

[0086] In some embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1) at a temperature above 30°C, or above or equal to 35°C, or above or equal to 40°C, or above or equal to 45°C, or above or equal to 50°C. The temperature is typically below 60°C. When the temperature is above 30°C, the duration of the crosslinking step is at least 1 minute, preferably at least 10 minutes, and even more preferably at least 1 hour, preferably between 1 and 5 hours.

[0087] In some embodiments, step (a2) can be carried out by placing the reaction medium directly obtained at the end of step (a1) at a pressure P less than or equal to atmospheric pressure and at a temperature T higher than the eutectic point temperature of the reaction medium as measured at pressure P and lower than the freezing point temperature of the reaction medium as measured at pressure P, preferably for a period of at least 1 hour. Crosslinked polysaccharide-based hydrogels prepared by such a process are highly biocompatible. Indeed, crosslinked polysaccharides can be prepared with smaller amounts of crosslinking agent, for example, amounts ranging from 0.001 to 0.02 moles. for 1 mole of repeating unit of the polysaccharide.

[0088] The freezing point temperature of the reaction medium refers to the temperature at which the mixture of reaction medium components, on a macroscopic scale, solidifies, that is, 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. This frozen state is maintained until the eutectic point temperature of the reaction medium is reached.

[0089] The eutectic point temperature of the reaction medium is the temperature below which the mixture of the reaction medium components transitions from a frozen state (coexistence of liquid and solid phases) to a completely solid state, that is, a state in which all the components of the mixture are in solid form. The freezing point and the eutectic point of a mixture depend on the pressure to which the mixture is subjected; therefore, the freezing point and the eutectic point are measured at pressure P.

[0090] The freezing point and the eutectic point can be determined by differential scanning calorimetry. This method makes it possible to determine phase transitions. For this purpose, the product to be studied is progressively cooled until its phase transitions are observed.

[0091] The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably ranging from -35°C to -10°C. Even more preferably, the temperature T is about -20°C.

[0092] The pressure P is preferably atmospheric pressure. "Atmospheric pressure" is the pressure exerted by the air constituting the atmosphere on any surface in contact with it. It varies with altitude. At an altitude of 0 m, the average atmospheric pressure is 101,325 Pa. 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 T varies from -35°C to -10°C or is approximately -20°C.

[0093] Preferably, during the crosslinking step (a2), when the temperature T is greater than or equal to -55°C and less than or equal to -5°C, the reaction medium obtained at the end of step (1) is placed for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably at most 27 weeks. Preferably, the crosslinking step (a2) is carried out for a period of 2 to 25 weeks, preferably from 2 to 20 weeks or 2 to 17 weeks, even more preferably from 3 to 8 weeks or 4 to 7 weeks, and at temperature T and pressure P.

[0094] At the end of step (a2), the cross-linked polysaccharide is typically in gel form. This gel is generally directly involved in the next step of the process. the invention (step (1)).

[0095] The crosslinked and / or non-crosslinked polysaccharides described above are useful for implementing the processes of the invention and thus preparing hydrogels comprising a crosslinked and / or non-crosslinked polysaccharide. The crosslinked or non-crosslinked polysaccharide, or a mixture thereof, will constitute the polymer network of the hydrogel. The hydrogel comprising a crosslinked or non-crosslinked polysaccharide, or a mixture thereof, can thus be said to be based on a crosslinked polysaccharide, a non-crosslinked polysaccharide, or a mixture thereof. A hydrogel comprising, as its sole polysaccharide, a non-crosslinked polysaccharide, is prepared from a non-crosslinked polysaccharide. A hydrogel comprising, as its sole polysaccharide, a crosslinked polysaccharide, is prepared from a crosslinked polysaccharide. When the hydrogel comprises a mixture of a cross-linked and a non-cross-linked polysaccharide, the hydrogel is prepared from a cross-linked polysaccharide and a non-cross-linked polysaccharide.The non-crosslinked polysaccharide is typically added to the crosslinked polysaccharide during the preparation of the hydrogel. Preparation of the hydrogel (step (1))

[0096] The process of the present invention according to method 1 comprises the preparation of a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide.

[0097] The preparation of the hydrogel comprises the following steps:

[0098] - contacting the cross-linked and / or non-cross-linked polysaccharide with a physicochemical solution saline physiological solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0099] - addition of zinc ions and citrates to the cross-linked and / or non-cross-linked polysaccharide in a molar ratio of citrate ions / zinc ions ranging from 1 to 20 and in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel and a zinc ion concentration of at most 20 mM in the hydrogel,

[0100] provided that the addition of zinc ions is not carried out before the addition of citrate ions when the contact of the crosslinked and / or non-crosslinked polysaccharide with the physiological saline solution, preferably buffered, is carried out before the addition of citrate ions.

[0101] When physiological saline is buffered, it is buffered with phosphate and / or carbonate and / or sulfate salts and generally has a physiological pH (6.8-7.8). Generally, the buffered physiological saline is a saline solution containing phosphate salts; preferably, the buffered saline solution can be a phosphate buffer. The phosphate buffer can be a PBS buffer with a pH around physiological pH (6.8-7.8) (CAS No: 7647-14-5, 7447-40-7). Preferably, the buffer is a phosphate, particularly a saline buffer of NahhPCVNaoHPChou of KH2PO4 / K2HPO4.

[0102] During the preparation of a hydrogel, contacting of the crosslinked and / or non-crosslinked polysaccharide with a physiological saline solution, preferably buffered, comprising phosphate, carbonate, or sulfate salts, or mixtures thereof, can occur at various stages. For example, contacting of the crosslinked and / or non-crosslinked polysaccharide with the physiological saline solution, preferably buffered, can occur when adjusting the concentration of the crosslinked and / or non-crosslinked polysaccharide in the prepared hydrogel. This step is commonly referred to as "dilution." Thus, during the preparation of the hydrogel, a physiological saline solution, preferably buffered, comprising phosphate, carbonate, or sulfate salts, or mixtures thereof, is typically added during the dilution stage.Alternatively or in addition, the contact of the cross-linked and / or non-cross-linked polysaccharide with physiological saline solution, preferably buffered, can occur at the time of pH adjustment or at the time of the addition of one or more additional components (see below).

[0103] Preferably, in the process of the present invention, contact with the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof, is made at least during the step of adjusting the concentration of crosslinked and / or non-crosslinked polysaccharide (dilution).

[0104] The amount of citrate ions added allows a concentration of citrate ions in the hydrogel to be achieved of at least 0.1 mM. Preferably, the amount of citrate ions added allows a concentration of citrate ions in the hydrogel to be achieved ranging from 0.1 to 20 mM or from 0.1 to 15 mM or from 0.3 to 15 mM or from 0.5 to 15 mM or from 1 to 10 mM or from 1.5 to 10 mM or from 2 to 10 mM or from 3 to 10 mM or from 3 to 8 mM or from 5 to 8 mM.

[0105] The amount of zinc ions added allows a concentration of zinc ions in the hydrogel not exceeding 20 mM, or not exceeding 7 mM, or not exceeding 5 mM, or not exceeding 3.5 mM, or not exceeding 2 mM, or not exceeding 1.6 mM, or not exceeding 1.15 mM, or not exceeding 0.8 mM. The amount of zinc ions added allows a zinc ion concentration in the hydrogel to be achieved, typically ranging from 0.10 to 20 mM or from 0.10 to 7 mM or from 0.10 to 5 mM or from 0.10 to 3.5 mM or from 0.10 to 2 mM or from 0.10 to 1.6 mM, or from 0.10 to 1.15 mM or from 0.10 to 0.8 mM. In some embodiments, the amount of zinc ions added allows a zinc ion concentration in the hydrogel to be achieved ranging from 0.10 to 1.6 mM, more preferably from 0.10 to 1.15 mM, even more preferably from 0.1 to 0.8 mM, preferably from 0.3 to 0.8 mM.

[0106] The molar ratio of citrate ions / added zinc varies from 1 to 20, preferably from 5 to 20 or from 5 to 15 or from 6 to 15 or from 6 to 10.

[0107] Citrate and zinc ions may be added before or after contacting the crosslinked and / or non-crosslinked polysaccharide with physiological saline, preferably buffered, containing phosphate, carbonate, or sulfate salts, or mixtures thereof. The citrate and zinc ions may be added sequentially, either before or after contacting the crosslinked and / or non-crosslinked polysaccharide with physiological saline, preferably buffered. For example, citrate ions may be added first, then zinc ions, or zinc ions may be added first, then citrate ions. However, the addition of zinc ions before the addition of citrate ions is only permissible if the addition of citrate and zinc ions is carried out before contacting the crosslinked and / or non-crosslinked polysaccharide with physiological saline, preferably buffered.Alternatively, citrate and zinc ions can be added concomitantly, either before or after the step of contacting the crosslinked and / or non-crosslinked polysaccharide with physiological saline solution, preferably buffered.

[0108] In other embodiments, the addition of citrate and zinc ions is concomitant with the contacting of the crosslinked and / or non-crosslinked polysaccharide with physiological saline, preferably buffered. In this case, the physiological saline, preferably buffered, may comprise phosphate and / or carbonate and / or sulfate salts or mixtures thereof, and further comprise citrate and zinc ions.

[0109] Citrate ions can be added in powder form or as a solution. The solution can be prepared by adding citric acid to water or to a physiological saline solution, preferably buffered, for example to a buffered physiological saline solution comprising phosphate or carbonate or sulfate salts or mixtures thereof.

[0110] Zinc ions can be added in powder form or as a solution. The solution can be prepared by adding zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate to water.

[0111] Preferably, the zinc and citrate ions can be added as a solution comprising zinc and citrate ions. A solution comprising both citrate and zinc ions is then added during the preparation of the hydrogel. The solution can be prepared by adding citric acid to water and then adding zinc salts (e.g., zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate), preferably by adding zinc chloride. Alternatively, the solution can be prepared by adding citric acid to A physiological saline solution, preferably buffered, is prepared, followed by the addition of zinc salts (e.g., zinc acetate and / or zinc chloride and / or zinc sulfate and / or zinc oxide and / or zinc gluconate). Preferably, the zinc salt is zinc chloride. The buffered physiological solution may be, for example, a phosphate buffer. The phosphate buffer may be a PBS buffer with a pH around physiological pH (6.8–7.8) (CAS No.: 7647-14-5, 7447-40-7). Preferably, the buffer is a phosphate buffer, particularly a saline buffer of NaH₂PO₄ / Na₂HPO₄ or KH₂PO₄ / K₂HPO₄. If necessary, the pH of the solution may be adjusted to reach physiological pH (6.8–7.8), for example, by adding sodium hydroxide. Thus, in some embodiments, a phosphate buffer solution comprising zinc and citric acid, with a pH ranging from 6.8 to 7.8, is added during the preparation of the hydrogel.

[0112] The preparation of a hydrogel from the cross-linked and / or non-cross-linked polysaccharide can be carried out conventionally, with the difference that zinc ions and citrate ions are added during the preparation of the hydrogel. Thus, the preparation of a hydrogel from the cross-linked and / or non-cross-linked polysaccharide can include one or more of the following conventional steps: - Adjustment of the pH (1); - Dilution (2); - Purification (3); - Addition of at least one additional component (4); - Homogenization (5).

[0113] These steps, well known to a person skilled in the art, can be as described below.

[0114] These steps, well known to those skilled in the art, can be as described below. The conventional steps can be carried out concurrently. The conventional steps can be carried out sequentially as follows: possible pH adjustment (1), then possible dilution (2), then possible purification (3), then possible addition of an additional component (4), then possible homogenization (5). They can also be carried out in a different order. Advantageously, the homogenization step (5) is carried out last, when at least one of the other conventional steps has been implemented. It can also be carried out repeatedly and interspersed between the other conventional steps described.

[0115] For example, conventional steps can be carried out in the following sequential manner: (1), (2), (3), (4), (5); or (2), (1), (3), (4), (5); or (2), (1), (4), (5); or (2), (4), (5); or (1), (4), (5); or (2), (4), (3), (5); or (2), (4), (1), (5); or (2), (4), (5); or (4), (2), (1); or (4), (1), (2) or (2), (3), (4), (5); or (4), (2), (3), (5); or (2), (4), (1); or (1), (5), (3), (4); or (1), (5), (4); or (2), (4). Steps (2), (3), (4) and (5) steps (2) and (4) can be concurrent. For example, the preparation of the hydrogel may include the following sequence: (2) and (4) are carried out concurrently.

[0116] Citrate ions (in powder or solution form) and zinc ions (in powder or solution form) can be added at, before, or after any of these conventional steps. Typically, when citrate ions are added in powder form, the effect of the citrate ions on the pH of the hydrogel can be neutralized. Citrate and zinc ions are preferably added as a solution comprising zinc and citrate ions. The solution is as described above.

[0117] Citrate ions (in powder form or in solution) and / or zinc ions (in powder form or in solution) are preferably added during step (2) or (4), preferably during step (4). Steps (2) and (4) can be carried out concurrently.

[0118] In one embodiment, citrate ions and / or zinc ions are added before the homogenization step (5) so as to obtain a homogeneous gel.

[0119] When a purification step (3) is implemented, the citrate ions and / or zinc ions are advantageously added after the purification step (3). The addition of the citrate ions and / or zinc ions after the purification step ensures better control of the concentration of citrate ions and / or zinc ions in the prepared hydrogel.

[0120] Preferably, citrate ions and / or zinc ions are added between the purification (3) and homogenization (5) steps.

[0121] The addition of citrate and zinc ions can be carried out after the dilution step (2) or during the dilution step (2), for example the citrate ions can be added to the aqueous dilution solvent.

[0122] Preferably, citrate ions in powder form or in solution (advantageously the solution comprising citrate ions) and / or zinc ions are added during the dilution step (2) and / or during the step of adding at least one additional component (4), preferably during the step of adding at least one additional component (4). In particular, in some embodiments, the addition of citrate ions and / or zinc ions (advantageously the solution comprising citrate and zinc ions) is concomitant with the step of adding at least one additional component (4).

[0123] Preferably, citrate ions in powder form or in solution (advantageously the solution comprising citrate ions) and / or zinc ions are added during the dilution step (2) and / or during the step of adding at least one additional component (4), preferably during the step of adding at least one additional component (4). In particular, in some embodiments, the addition of citrate ions and / or zinc ions (advantageously the solution comprising citrate and zinc ions) is concomitant with the step of adding at least an additional component (4).

[0124] In particular, in certain embodiments, the addition of zinc ions and citrate ions, preferably in the form of a solution comprising citrate and zinc ions, is concomitant with the addition of an anesthetic solution.

[0125] In particular, in certain embodiments, the addition of zinc ions and citrate ions, preferably in the form of a solution comprising citrate and zinc ions, is concomitant with the addition of a lubricating agent.

[0126] In some embodiments, the added solution comprising zinc ions and citrate ions may include other components, in particular a lubricating agent, for example non-crosslinked hyaluronic acid, non-crosslinked heparosan or a mixture thereof.

[0127] The steps of dilution (2), addition of at least one additional component (4) and addition of citrate and / or zinc ions may be concomitant.

[0128] Citrate and / or zinc ions can be added after the pH adjustment step (1). Citrate ions can be added between the pH adjustment step (1) and the homogenization step (5) when both of these steps are carried out. pH adjustment (1)

[0129] The hydrogel preparation process may include a step of adjusting the pH of the hydrogel to achieve the desired pH (pH of 6.8-7.8). Dilution (2)

[0130] The hydrogel preparation process may include a dilution step of the crosslinked and / or non-crosslinked polysaccharide. The dilution step allows the concentration of crosslinked and / or non-crosslinked polysaccharide in the prepared hydrogel to be adjusted. In particular, an aqueous solvent is added to the crosslinked and / or non-crosslinked polysaccharide, for example, a physiological saline solution, possibly buffered by the presence of salts, such as phosphate, carbonate, or sulfate salts, or mixtures thereof. More specifically, the added aqueous solvent has a pH around the physiological pH (6.8–7.8). The polysaccharide concentration obtained following the dilution step advantageously ranges from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, and even more advantageously from 10 mg / g to 30 mg / g of hydrogel. Purification (3)

[0131] The hydrogel preparation process may include at least one purification step. The purification step aims to remove any undesirable impurities. These undesirable impurities may result from the crosslinking of the polysaccharide, for example, from step (a2) described above. Such impurities may include, for example, residual crosslinking agent, in particular of the type epoxy, which would not have reacted. This step can also allow for a liquid exchange, for example a buffer exchange.

[0132] The purification step can therefore be implemented particularly when the hydrogel includes a cross-linked polysaccharide.

[0133] Purification can be carried out by dialysis or by filtration, for example by dynamic cross-flow filtration (DCF). Addition of additional components (4)

[0134] The hydrogel preparation process may include a step of adding at least one additional component. The additional component may be selected from anesthetic agents, antioxidants, lubricating agents, amino acids, peptides, proteins, vitamins, minerals, nucleic acids, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate monohydrate and / or dihydrate, sodium chloride and a mixture thereof.

[0135] Non-crosslinked polysaccharides, in particular non-crosslinked hyaluronic acid, non-crosslinked heparosan or mixtures thereof, may be cited as examples of lubricating agents.

[0136] Examples of anesthetics include, but are not limited to, Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Beta-toxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chloro-robutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryo-fluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuverine, Fomocaine, Guafecaine, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamide, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, Myrtecaine, Octacaine, Octodrine, Oxetacaine, Oxybuprocaine, Parethoxycaine, Paridocaine, Phenacaine, Piperocaine, Piridocaine, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, ProxymetacainePyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and one of their salts, in particular a hydrochloride salt, or a mixture thereof. Preferably, the hydrogel according to the invention comprises an anesthetic agent as defined above, and in particular lidocaine, mepivacaine, or one of their salts, such as the hydrochloride.

[0137] 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 of these.

[0138] Examples of amino acids include, but are not limited to, arginine (e.g., L-arginine), isoleucine (e.g., L-isoleucine), leucine (e.g., L-leucine), lysine (e.g., L-lysine or L-lysine monohydrate), glycine, valine (e.g., L-valine), threonine (e.g., L-threonine), proline (e.g., L-proline), methionine, histidine, phenylalanine, tryptophan, cysteine, their derivatives (e.g., N-acetylated derivatives such as N-acetyl-L-cysteine) and mixtures thereof.

[0139] 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 preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0140] Examples of minerals include, but are not limited to, zinc salts (e.g. zinc acetate, especially dehydrated), magnesium salts, calcium salts (e.g. hydroxyapatite, especially in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g. copper sulfate, especially pentahydrate), possibly in hydrated form, and mixtures thereof.

[0141] Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymidine, cytodine, their derivatives, and mixtures thereof. Examples of coenzymes include coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

[0142] Adrenaline, noradrenaline, and mixtures thereof may be cited as derivatives of adrenaline. Homogenization (5)

[0143] The hydrogel preparation process may include one or more homogenization steps. This homogenization step makes it possible to obtain a more homogeneous hydrogel, in particular with the most constant, i.e., the most regular, extrusion force possible. For example, the homogenization step may consist of an extrusion step, more particularly using a sieve with perforations having a diameter between 50 and 2000 µm. Those skilled in the art know how to select the perforation diameter according to the desired mechanical properties of the hydrogel. Sterilization of the hydrogel (step (2))

[0144] The process of the present invention includes a step of sterilizing the prepared hydrogel. Sterilization is preferably carried out by heat, for example in an autoclave. Sterilization is generally performed by increasing the temperature of the sterilization medium to a temperature known as the "plateau temperature," which is Sterilization is maintained for a specific duration, known as the "plateau time." It is preferably carried out at a plateau temperature ranging from 121°C to 135°C, and preferably at a plateau time ranging from 1 to 20 minutes with a FO value greater than 15. The FO sterilization value corresponds to the time required, in minutes, at 121°C, to inactivate 90% of the microorganism population present in the product to be sterilized. Alternatively, sterilization can be carried out by gamma radiation, UV radiation, or using ethylene oxide.

[0145] The hydrogel obtained at the end of the process typically has a pH ranging from 6.8 to 7.8 (physiological pH). Method 2

[0146] The present invention also relates to a method for preparing a sterile hydrogel comprising a cross-linked polysaccharide and optionally a non-cross-linked polysaccharide, and further comprising zinc ions, the method comprising the following steps:

[0147] (0) preparation of a crosslinked polysaccharide from a reticulated reaction medium culation comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and zinc ions in an amount permitting the preparation of a hydrogel comprising at most 20 mM of zinc ions;

[0148] (1) preparation of a hydrogel from the cross-linked polysaccharide obtained at the end of step (0) and optionally of a non-crosslinked polysaccharide, the preparation of the hydrogel comprising contacting the crosslinked polysaccharide with a physiological saline solution, preferably buffered, the physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;

[0149] (2) sterilization, preferably by heat, of the hydrogel obtained at the end of the step (1) to obtain a sterile hydrogel;

[0150] in which:

[0151] - the crosslinking reaction medium further comprises citrate ions in a sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, with a molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0152] - step (1) further comprises, before contacting the cross-linked polysaccharide with physiological saline solution, preferably buffered, a step of adding citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions added] / [zinc ions present in the reaction medium] ranging from 1 to 20; or

[0153] - the physiological saline solution, preferably buffered, further comprises citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] ranging from 1 to 20.

[0154] The cross-linked polysaccharide can in particular be prepared by a process comprising the following steps:

[0155] (al) prepare a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent, zinc ions in an amount sufficient to prepare a hydrogel comprising at most 20 mM zinc ions and optionally citrate ions in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel; and

[0156] (a2) react the reaction medium to obtain a cross-linked polysaccharide.

[0157] Steps (0), (a1), and (a2) of the process according to Method 2 are as described previously in the section "Cross-linked and / or non-cross-linked polysaccharide," except that the cross-linking reaction medium further comprises zinc ions and optionally citrate ions. When citrate ions are not present in the cross-linking reaction medium, they are added before the step of contacting the cross-linked polysaccharide with physiological saline, preferably buffered, or into physiological saline, preferably buffered. The polysaccharide is as described in the section "Cross-linked and / or non-cross-linked polysaccharide."

[0158] Zinc ions are present in the reaction medium in an amount that does not exceed a zinc ion concentration in the hydrogel of 20 mM, or 7 mM, or 5 mM, or 3.5 mM, or 2 mM, or 1.6 mM, or 1.15 mM, or 0.8 mM. Zinc ions are typically present in the reaction medium in an amount that allows a zinc ion concentration in the hydrogel to be achieved that varies from 0.10 to 20 mM, or from 0.10 to 7 mM, or from 0.10 to 5 mM, or from 0.10 to 3.5 mM, or from 0.10 to 2 mM, or from 0.10 to 1.6 mM, or from 0.10 to 1.15 mM, or from 0.10 to 0.8 mM. In some embodiments, zinc ions are present in the reaction medium in an amount that allows a zinc ion concentration in the hydrogel to be achieved ranging from 0.10 to 1.6 mM, more preferably from 0.10 to 1.15 mM, even more preferably from 0.1 to 0.8 mM, preferably from 0.3 to 0.8 mM.

[0159] When citrate ions are present in the reaction medium, they are present in an amount that allows a citrate ion concentration in the hydrogel to be at least 0.1 mM and generally at most 20 mM or 15 mM. Preferably, they are present in an amount that allows a citrate ion concentration in the hydrogel to be reached that varies from 0.1 to 15 mM or from 0.3 to 15 mM or from 0.5 to 15 mM or from 1 to 10 mM or from 1.5 to 10 mM or from 2 to 10 mM or from 3 to 10 mM or from 3 to 8 mM or from 5 to 8 mM.

[0160] When citrate ions are not present in the reaction medium, they are added before contact with physiological saline solution, preferably buffered, or in physiological saline solution, preferably buffered, in sufficient quantity to reach the concentrations indicated above.

[0161] The molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] or [added citrate ions] / [zinc ions present in the reaction medium] or [citrate ions present in physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] varies from 1 to 20, preferably from 5 to 20 or from 5 to 15 or from 6 to 15 or from 6 to 10.

[0162] The zinc ions present in the reaction medium may result from the addition of zinc salts to the reaction medium such as zinc sulfate, zinc chloride, zinc gluconate, preferably zinc chloride.

[0163] The citrate ions present in the reaction medium may result from the addition of citric acid, in powder form or as an aqueous solution, to the reaction medium.

[0164] When citrate ions are not present in the reaction medium, they are added before the step of contacting the physiological saline solution, preferably buffered, or into the physiological saline solution, preferably buffered. They may be added as a solution. The solution may be prepared by adding citric acid to water or to a physiological saline solution, preferably buffered, for example, a saline solution, preferably buffered, comprising phosphate, carbonate, or sulfate salts, or mixtures thereof. In other embodiments, the physiological saline solution, preferably buffered, added during the preparation of the hydrogel comprises citrate ions.

[0165] At the end of step (0) or (a2), the cross-linked polysaccharide is typically in the form of a gel comprising zinc ions, and possibly citrate ions. This gel is generally directly involved in the continuation of the process of the invention (step (D).

[0166] The preparation of a hydrogel (step (1)) from the cross-linked polysaccharide obtained at the end of step (0) or (a2) can be carried out conventionally. In particular, the preparation of a hydrogel from the cross-linked polysaccharide obtained at the end of step (0) or (a2) typically comprises one or more of the following conventional steps: - pH adjustment (1); - Dilution (2); - Purification (3); - Addition of at least one additional component (4); - Homogenization (5).

[0167] These steps, well known to those skilled in the art, can be as described above in relation to method 1. They can be implemented in the sequential ways described above.

[0168] Sterilization (step (2)) is as described in relation to step (2) of method 1. Method 1 or 2: Optional step

[0169] The process of the present invention (method 1 or 2) may further include a step of conditioning the hydrogel. The conditioning of the hydrogel is typically carried out in an injection device. The conditioning is preferably carried out just before the sterilization step (step (2)). Thus, the sterile hydrogel may be in the form of an injection device pre-filled with the hydrogel, for example, a syringe pre-filled with the hydrogel. sterile hydrogel

[0170] The present invention also relates to a sterile hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide obtained or obtainable by the process of the present invention (method 1 or 2). The sterile hydrogel comprises zinc ions and citrate ions in a citrate / zinc molar ratio ranging from 1 to 20, the concentration of citrate ions in the hydrogel being at least 0.1 mM and the concentration of zinc in the hydrogel not exceeding 20 mM.

[0171] Preferably, the zinc ion concentration in the hydrogel varies from 0.10 to 20 mM or from 0.10 to 7 mM or from 0.10 to 5 mM or from 0.10 to 3.5 mM or from 0.10 to 2 mM or from 0.10 to 1.6 mM, or from 0.10 to 1.15 mM or from 0.10 to 0.8 mM. In some embodiments, the zinc ion concentration in the hydrogel varies from 0.10 to 1.6 mM, more preferably from 0.10 to 1.15 mM, even more preferably from 0.1 to 0.8 mM, preferably from 0.3 to 0.8 mM.

[0172] Preferably, the concentration of citrate ions in the hydrogel varies from 0.1 to 20 mM or from 0.1 to 15 mM or 0.3 to 15 mM or from 0.5 to 15 mM or from 1 to 10 mM or from 1.5 to 10 mM or from 2 to 10 mM or from 3 to 10 mM or from 3 to 8 mM or from 5 to 8 mM.

[0173] The sterile hydrogel obtained or capable of being obtained by the process of the present invention (method 1 or 2) has a physiological pH, i.e., ranging from 6.8 to 7.8. The pH of the sterile hydrogel is preferably greater than or equal to 6.9 and less than or equal to 7.4; 7.3; 7.2; 7.1 or 7.

[0174] The sterile hydrogel obtained by the process of the present invention (method 1 or 2) and comprising a cross-linked polysaccharide, advantageously has a phase angle θ less than or equal to 45°, at 1Hz for a strain of 0.1% or a pressure of 1 Pa, preferably a phase angle θ ranging from 2° to 45° or from 20° to 45°.

[0175] The hydrogel obtained or capable of being obtained by the process of this The invention is preferably an injectable hydrogel, that is to say, one which can flow and be manually injected using a syringe fitted with a needle with a diameter of 0.1 to 0.5 mm, for example a 32G, 30G, 27G, 26G, 25G hypodermic needle.

[0176] The hydrogel obtained or obtainable by the process of the present invention may comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of polysaccharide (total weight of polysaccharide, i.e., total weight of cross-linked and / or non-cross-linked polysaccharide, for example, cross-linked and / or non-cross-linked hyaluronic acid), relative to the total weight of the hydrogel. Thus, when the hydrogel comprises, as its sole polysaccharide, a non-cross-linked polysaccharide, the hydrogel obtained by the process of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of non-cross-linked polysaccharide (for example, non-cross-linked hyaluronic acid), relative to the total weight of the hydrogel.When the hydrogel comprises, as its sole polysaccharide, a cross-linked polysaccharide, the hydrogel obtained by the process of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of cross-linked polysaccharide (for example, cross-linked hyaluronic acid), relative to the total weight of the hydrogel. When the hydrogel comprises a mixture of a cross-linked and non-cross-linked polysaccharide, the hydrogel obtained by the process of the present invention may therefore comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of a mixture of non-cross-linked and cross-linked polysaccharide (for example, non-cross-linked and / or cross-linked hyaluronic acid), relative to the total weight of the hydrogel.In particular, the content of non-crosslinked polysaccharide (e.g. hyaluronic acid) can vary from 0.5 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 (e.g. hyaluronic acid) present in the hydrogel.

[0177] The total polysaccharide concentration in the hydrogel obtained by the process of the present invention advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, and even more advantageously from 10 mg / g to 30 mg / g of hydrogel. Preferably, the polysaccharide is hyaluronic acid, and even more preferably, sodium hyaluronate.

[0178] The total polysaccharide concentration in the hydrogel obtained by the process of the present invention advantageously varies from 1 mg / g to 50 mg / g of hydrogel, more advantageously from 5 mg / g to 35 mg / g of hydrogel, and even more advantageously from 10 mg / g to 30 mg / g of hydrogel. Preferably, the polysaccharide is hyaluronic acid, and even more preferably, sodium hyaluronate.

[0179] When the hydrogel comprises a cross-linked polysaccharide, the cross-linked polysaccharide preferably has a molar cross-linking ratio of 10% or less. Preferably Preferably, the hydrogel comprises a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 6%. Even more preferably, the hydrogel comprises a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 4%. Even more preferably, the hydrogel comprises a crosslinked polysaccharide with a molar crosslinking ratio greater than 0 and less than or equal to 2%, preferably less than or equal to 1%, and even more preferably less than or equal to 0.8%, in particular ranging from 0.1% to 0.5% (number of moles of crosslinking agent(s) per 100 moles of repeating unit(s) of the polysaccharide(s)).

[0180] When the hydrogel comprises a cross-linked polysaccharide, the cross-linked polysaccharide preferably has a degree of modification (MOD) of 10% or less, preferably 6% or less, preferably 4% or less, preferably 2% or less, and more preferably 1% or less. Advantageously, the cross-linked polysaccharide has a degree of modification (MOD) of 1.8% or less, more preferably 1.5% or less, preferably 1.2% or less, and even more preferably less than 1%.

[0181] In some embodiments, the hydrogel comprises an anesthetic agent. The anesthetic agent may be as described above, in particular the anesthetic agent may be mepivacaine, lidocaine or one of their salts; more particularly in the form of a hydrochloride salt; preferably in amounts from 0.1 to 30 mg / ml, for example from 0.5 to 10 mg / ml or more preferably from 2 to 6 mg / ml.

[0182] Sterile hydrogels prepared according to the process of the invention are particularly useful for filling and / or replacing tissues, especially soft tissues, notably by injecting the hydrogel into the tissue. In addition to filling soft tissues, they provide biostimulating effects.

[0183] They can be injected using any of the methods known to those skilled in the art. In particular, they can be administered using an injection device suitable for intra-epidermal and / or intradermal and / or subcutaneous and / or supraperiosteal injection. The injection device can, in particular, be selected from a syringe, a set of microsyringes, a thread, a laser or hydraulic device, an injection gun, a needle-free injection device, or a microneedle roller.

[0184] Sterile hydrogels prepared according to the process of the invention are preferably injected subcutaneously.

[0185] They may relate to deep applications, mid-level applications and / or shallow applications.

[0186] They may have therapeutic and / or cosmetic and / or cosmé- applications ceutiques.

[0187] In the cosmetic field, hydrogels can be particularly useful for compensating for tissue volume losses due to aging.

[0188] They can be used in the prevention and / or cosmetic treatment of alterations in the skin's surface appearance. For example, hydrogels can be used in cosmetics to prevent and / or treat alterations in the viscoelastic or biomechanical properties of the skin; to fill volume defects in the skin, in particular to fill wrinkles, fine lines and scars; to reduce nasolabial folds and marionette lines; to increase the volume of the cheekbones, chin or lips; to restore facial volume, in particular of the cheeks, temples, jawline and around the eyes; to reduce the appearance of wrinkles and fine lines.

[0189] The present invention also relates to the cosmetic use of a hydrogel as described above for filling tissues, in particular soft tissues, in particular to compensate for tissue volume losses due to aging.

[0190] The following examples are given by way of illustration, but shall in no way be considered as limiting the present invention. EXAMPLES 1. Materials

[0191] - Non-crosslinked sodium hyaluronate

[0192] - BDDE (Sigma Aldrich)

[0193] - Citric acid (Sigma Aldrich)

[0194] - Zinc chloride (Sigma Aldrich)

[0195] - Commercial zinc citrate (CAS 5990-32-9 - Thermo Fisher)

[0196] - NaOH 0.25M

[0197] - HCl IM

[0198] - PBS Phosphate Buffer (BBraun),

[0199] - Lidocaine Hydrochloride

[0200] - Three-dimensional agitator

[0201] - DHR-2 Rheometer

[0202] - Dynamometer and test bench

[0203] - Homogenizer Paddle Mill

[0204] - Sterile polyethylene bag 2. Methods Measurement of viscoelastic properties

[0205] The viscoelastic properties of the hydrogels obtained were measured using a rheometer (DHR-2) having a stainless steel cone (1° - 40 mm) with cone- geometry plan and an anodized aluminum peltier plan (42 mm) (air gap 24 pm).

[0206] 0.5 g of sterilized hydrogel is deposited between the Peltier plane and said cone. Then a Stress scanning is performed at 1 Hz and 25°C. The elastic modulus G', viscous modulus G” and phase angle θ are recorded for a stress of 5 Pa. The measurements are carried out in the linear LVER domain.

[0207] The constraint at the intersection of G' and G”, r, is determined at the intersection of the curves of the modules G' and G'' and is expressed in Pascal. 3. Examples 3.1 Example the

[0208] A cross-linked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid (4 MDa) and BDDE in a 0.25 M aqueous sodium hydroxide solution. The cross-linked polysaccharide has a cross-linking ratio of 2% and a hyaluronic acid concentration of 15 mg per gram of hydrogel. Phosphate buffer and HCl₂IN solution are then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The resulting hydrogel is homogenized using a three-dimensional stirrer. The hydrogel is then dialyzed.

[0209] The following are then added to the hydrogels obtained:

[0210] - a high molecular weight sodium hyaluronate solution as a brisant (same quantity in the different mixtures);

[0211] - an aqueous solution of lidocaine hydrochloride to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel;

[0212] - possibly a solution comprising zinc and citrate ions.

[0213] The solution containing zinc and citrate ions is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride (ZnCl2 powder) is dissolved, and finally 5M NaOH is added to adjust the pH to a physiological level. The aim is to make a solution that is 100 times more concentrated (in zinc and citrate ions) than the actual concentration required in the final hydrogel. This prevents excessive dilution of the hydrogel due to the addition of the zinc and citric acid solution.

[0214] The solution comprising zinc and citrate ions is added at the same time as the anesthetic solution, after the addition of the high molecular weight sodium hyaluronate solution.

[0215] The hydrogels obtained were sieved and then packaged in syringes.

[0216] Finally, the hydrogels obtained were sterilized by autoclave (temperature at the plateau between 121°C and 135°C with F0 > 15).

[0217] After sterilization, the hydrogels A1-A6 were analyzed. No hydrogel exhibited precipitation. The elastic modulus G' and the phase angle θ were determined. completed. The results are presented in Table 1 below.

[0218] The hydrogels exhibit a molar crosslinking rate of 2%.

[0219] [Table 1] Hydrogels Al A2 A3 A4 A5 A6 Molar concentration of citrate ions in final hydrogel (mM) 0.00 0.77 2.30 3.83 5.74 7.65 Mass percentage of citrate ions in final hydrogel (%w / w) 0.00 0.02 0.05 0.08 0.12 0.16 Molar concentration of zinc in final hydrogel (mM) 0.00 0.15 0.46 0.77 1.15 1.53 Mass percentage of zinc in final hydrogel (%w / w) 0.00 0.0010 0.0030 0 0.0050 0.0075 0.0100 G' (1 Hz) before sterilization 127.7 125.8 129.1 125.5 124.6 123.3 Δ before sterilization 14.0 14.8 13.6 14.9 15.3 15.5 AG' (%)' -40.0 -36.5 -29.2 -22.5 -23.2 -18.2 A Δ (%)2 50.3 42.2 34.8 27.0 26 22.2

[0220] Table 1

[0221] 'AG' (%)= (G' after sterilization - G' before sterilization) / (G' before sterilization) *100

[0222] 2 A ô (%)= (ô after sterilization - ô before sterilization) / ( ô before sterilization) *100

[0223] It is observed that hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising zinc ions and citrate ions exhibit less modification of their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without the addition of such a solution. 3.2 Example 1b

[0224] A cross-linked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid 4MDa and BDDE in a 0.25M aqueous sodium hydroxide solution. The cross-linked polysaccharide has a modification rate of 2% and a concentration of 15 mg of hyaluronic acid per gram of product. Buffer Phosphate (PBS) and a solution of HCl (IN) are then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The resulting hydrogel is homogenized using a three-dimensional stirrer. The hydrogel is then dialyzed.

[0225] The following are then added to the hydrogels obtained:

[0226] - a non-crosslinked high molecular weight sodium hyaluronate solution as a lubricant (same quantity in the different mixtures);

[0227] - possibly a solution comprising zinc and citrate ions.

[0228] The solution containing zinc and citrate ions is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride (ZnCl2 powder) is dissolved, and finally 5M NaOH is added to adjust the pH to a physiological level. The aim is to make a solution that is 100 times more concentrated (in zinc and citrate ions) than the actual concentration required in the final hydrogel. This prevents excessive dilution of the hydrogel due to the addition of the zinc and citric acid solution.

[0229] The solution comprising zinc and citrate ions is added after the addition of the high molecular weight sodium hyaluronate solution.

[0230] The hydrogels obtained were sieved and then packaged in syringes.

[0231] Finally, the hydrogels obtained are sterilized by autoclave (temperature at the tray between 121°C and 135°C with F0 > 15).

[0232] After sterilization, the B1-B4 hydrogels were analyzed. No hydrogel exhibited precipitation. The elastic modulus G', the phase angle θ, and the stress at the intersection of G' and G”, r were determined. The results are presented in Table 2 below.

[0233] The hydrogels exhibit a molar crosslinking rate of 2%.

[0234] [Tables2] Hydrogels B1 B2 B3 B4 Citric Acid Molar concentration of citrate ions in the final hydrogel (mM) 0.00 1.53 4.59 7.65 Mass percentage of citrate ions in the final hydrogel (%w / w) 0.00 0.03 0.10 0.16 Zinc Molar concentration of zinc in the final hydrogel (mM) 0.00 1.53 1.53 1.53 Mass percentage of zinc in the final hydrogel (%w / w) 0.00 0.01 0.01 0.01 G' (1 Hz) before sterilization 137.8 134.7 132.5 133.7 before sterilization 13.8 14.8 16.2 15.4 AG' (%)1 -40.8 -28.4 -24.6 -19.7 A ô (%)2 49.6 28.0 13.9 15.8

[0235] Table 2

[0236] 1 AG' (%)= (G' after sterilization - G' before sterilization) / (G' before sterilization) *100

[0237] 2 A ô (%)= (ô after sterilization - ô before sterilization) / ( ô before sterilization) *100

[0238] It is observed that hydrogels prepared from a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions exhibit less modification of their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without the addition of such a solution.

[0239] Furthermore, it has been observed that the addition of a solution comprising zinc and citrate ions helps to preserve the structure of hydrogels from degradation over time (Table 3).

[0240] [Tables3] Hydrogels B1 B4 G' (1 Hz) at T0 months 81.5 107.3 G' (1 Hz) T3 months 71.0 106.0 G' (1 Hz) T6 months 59.2 97.0 AG' (%)1 to 3 months -12.9 -1.2 AG' (%)2 to 6 months -27.4 -9.6

[0241] Table 3

[0242] 1 AG' (%) = (G' T3month - G' T0) / (G' T0) * 100

[0243] 2AG' (%) = (G' T6mois - G' T0) / (G' T0) *100

[0244] After 3 months and 6 months at 40°C, hydrogels prepared by a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions exhibit less change in their rheological properties after sterilization compared to hydrogels prepared by an equivalent process without the addition of such a solution. For hydrogel B4 prepared by a process according to the invention comprising a step of adding a solution comprising zinc and citrate ions, no precipitation is observed after either 3 or 6 months. 3.3 Example 2

[0245] For comparison, a hydrogel according to example 1b is made with the incorporation of 0.47g of commercial zinc citrate (in powder form, molar ratio of citrate ions / zinc ions in the hydrogel = 0.667) in buffer.

[0246] The solution containing commercial zinc citrate is prepared as follows. The zinc citrate is first dissolved in phosphate buffer and then added to 5M NaOH to adjust the pH to a physiological level. The aim is to make a solution that is 100 times concentrated (in zinc and citrate ions) relative to the actual concentration desired in the final hydrogel. This avoids an excessive dilution effect on the hydrogel due to the addition of the solution containing commercial zinc citrate.

[0247] The solution comprising commercial zinc citrate is added after the addition of the high molecular weight sodium hyaluronate solution.

[0248] The hydrogels obtained were sieved and then packaged in syringes.

[0249] Finally, the hydrogels obtained were sterilized by autoclave (temperature at the plateau between 121°C and 135°C with F0 > 15).

[0250] The results of the hydrogels observed under the microscope are presented in figures 1 and 2. The microscope used is: Olympus SZX16, the software: OLYMPUS Stream Start.

[0251] The hydrogel obtained according to the process of the invention ([Fig. 1] - prototype C3) is transparent.

[0252] A precipitate is observed immediately after sterilization of the hydrogel incorporating commercial zinc citrate ([Fig. 2]), which is undesirable for a hydrogel. 3.4 Example 3

[0253] A study conducted by the medical analysis laboratory NAMSA, according to the ISO 10993-23 standard "Biological Evaluation of Medical Devices, Part 23 (2021): Tests for Irritations", made it possible to evaluate the irritant potential of the hydrogels according to the invention after intradermal injection in rabbits.

[0254] The control hydrogels Cl and according to the invention C2 tested are prepared as follows:

[0255] The cross-linked hyaluronic acid hydrogel is prepared from a high molecular weight hyaluronic acid 1.5 MDa and BDDE in a 0.25 M aqueous sodium hydroxide solution. The cross-linked polysaccharide has a modification rate of 4% and a concentration of 23 mg of hyaluronic acid per gram of product. Phosphate buffer PBS and HCl IN solution are then added to the cross-linked polysaccharide until a pH of 7.3 ± 0.5 is obtained. The resulting hydrogel is homogenized using a three-dimensional stirrer. The hydrogel is then dialyzed.

[0256] The hydrogel based on a cross-linked polysaccharide obtained is subsequently divided into 2.

[0257] The following are then added to the hydrogels obtained:

[0258] - for the Cl and C2 hydrogel an aqueous solution of lidocaine hydrochloride for obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the final hydrogel and a non-crosslinked high molecular weight sodium hyaluronate solution as a lubricant (same quantity in the different mixtures);

[0259] - for the C2 hydrogel, a solution comprising 0.46 mM zinc ions and 2.3 mM of citrate ions.

[0260] The solution containing zinc and citrate ions is prepared as follows. Citric acid (in powder form) is first dissolved in phosphate buffer, then zinc chloride (ZnCl2 powder) is dissolved, and finally 5M NaOH is added to adjust the pH to a physiological level. The aim is to make a solution that is 100 times more concentrated (in zinc and citrate ions) than the actual concentration required in the final hydrogel. This prevents excessive dilution of the hydrogel due to the addition of the zinc and citric acid solution.

[0261] The solution comprising zinc and citrate ions is added after the addition of the high molecular weight sodium hyaluronate solution.

[0262] The hydrogels obtained were sieved and then packaged in syringes.

[0263] Finally, the hydrogels obtained are sterilized by autoclave (plate temperature between 121°C and 135°C with F0 > 15). No hydrogel shows precipitation.

[0264] A 0.2 mL dose of each of the sterilized hydrogels Cl and C2 is injected intra Dermal injections were performed at five separate sites on the sides of the backs of three rabbits. The injection sites were observed 24, 48, and 72 hours post-injection for signs of erythema and edema, and then daily for up to 28 days. Signs of erythema and edema were assessed using a 0-4 scoring scale for each injection site and for each animal. The overall mean score was determined by dividing the sum of the scores by the total number of sites assessed.

[0265] The hydrogel according to the invention C2 exhibits, throughout the test, a lower average irritation score than the control hydrogel Cl. This indicates that the hydrogel C2 prepared according to the invention is less irritating than the control hydrogel.

Claims

Demands

1. A process for preparing a sterile hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof, and further comprising zinc ions, the process comprising the following steps: (1) preparation of a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide, or a mixture thereof, the preparation of the hydrogel comprising the following steps: - contacting the cross-linked polysaccharide, the non-cross-linked polysaccharide, or a mixture thereof, with physiological saline, preferably buffered, the physiological saline, preferably buffered, comprising phosphate or carbonate or sulfate salts, or mixtures thereof, - adding citrate ions to the cross-linked polysaccharide, the non-cross-linked polysaccharide, or a mixture thereof, in an amount sufficient to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, - adding zinc ions to the cross-linked polysaccharide,to the non-crosslinked polysaccharide, or a mixture thereof, in sufficient quantity to achieve a zinc ion concentration of not more than 20 mM in the hydrogel, the addition of citrate ions and zinc ions being carried out in a molar ratio [citrate ions] / [zinc ions] ranging from 1 to 20, and provided that the addition of zinc ions is not carried out before the addition of citrate ions when contact with physiological saline, preferably buffered, is carried out before the addition of citrate ions; (2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof and further comprising zinc ions.

2. A method according to claim 1 wherein the addition of citrate ions and the addition of zinc ions are carried out after the step of contacting the physiological saline solution, preferably buffered.

3. A method according to claim 1 or 2 wherein the addition of citrate ions and the addition of zinc ions are carried out concomitantly, the citrate ions and the zinc ions being added in the form of a solution comprising zinc ions and citrate ions.

4. A method according to claim 3 wherein the solution is a solution physiological buffered saline, preferably a phosphate buffer, comprising zinc ions and citrate ions.

5. A process according to any one of claims 1 to 4 wherein step (1) of preparing a hydrogel comprises one or more of the following conventional steps: - pH adjustment; - Dilution; - Purification; - Addition of at least one additional component; - Homogenization.

6. A process for preparing a sterile hydrogel comprising a crosslinked polysaccharide and optionally a non-crosslinked polysaccharide and further comprising zinc ions, the process comprising the following steps: (0) preparing a crosslinked polysaccharide from a crosslinking reaction medium comprising one or more polysaccharide(s), one or more crosslinking agent(s), a solvent and zinc ions in an amount permitting the preparation of a hydrogel comprising at most 20 mM zinc ions; (1) preparing a hydrogel from the crosslinked polysaccharide obtained at the end of step (0) and optionally a non-crosslinked polysaccharide, the preparation of the hydrogel comprising a step of contacting the crosslinked polysaccharide with a physiological saline solution, preferably buffered, comprising phosphate or carbonate or sulfate salts or mixtures thereof;(2) sterilization, preferably by heat, of the hydrogel obtained at the end of step (1) to obtain a sterile hydrogel; wherein: - the crosslinking reaction medium further comprises citrate ions in sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the reaction medium] / [zinc ions present in the reaction medium] ranging from 1 to 20; or - step (1) further comprises, prior to the step of contacting the crosslinked polysaccharide with physiological saline solution, preferably buffered, a step of adding citrate ions in one; sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions added] / [zinc ions present in the reaction medium] ranging from 1 to 20; or - the physiological saline solution, preferably buffered, further comprises citrate ions in a sufficient quantity to achieve a citrate ion concentration of at least 0.1 mM in the hydrogel, the molar ratio [citrate ions present in the physiological saline solution, preferably buffered] / [zinc ions present in the reaction medium] ranging from 1 to 20.

7. A method according to any one of claims 1 to 6 wherein the polysaccharide is hyaluronic acid.

8. A method according to any one of claims 1 to 7 wherein the saline physiological solution is a buffered saline physiological solution comprising phosphate salts, preferably a phosphate buffer.

9. A method according to any one of claims 1 to 8 wherein step (1) further comprises a step of adding an anesthetic agent.

10. A method according to any one of claims 1 to 9 further comprising a step of conditioning the hydrogel, preferably in an injection device, after step (1) and before step (2).

11. A method according to any one of claims 1 to 10 wherein the sterilization is heat sterilization, preferably carried out in an autoclave.

12. Sterile hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, in particular a crosslinked hyaluronic acid, a non-crosslinked hyaluronic acid or a mixture thereof, and further comprising zinc and citrate ions obtained by the process according to any one of claims 1 to 11.

13. Hydrogel according to claim 12 further comprising an anesthetic agent.

14. Use of citrate ions to protect a hydrogel comprising a crosslinked polysaccharide, a non-crosslinked polysaccharide or a mixture thereof, optionally an anesthetic agent, and furthermore zinc ions, from the degradation of its rheological properties during sterilization, preferably by heat.

15. Use of citrate ions to preserve the stability over time of the rheological properties of a hydrogel comprising a polysaccharide crosslinked, a non-crosslinked polysaccharide or a mixture thereof, possibly an anesthetic agent, and in addition zinc ions.

16. Use of a solution comprising zinc ions and citrate ions to protect a hydrogel comprising a crosslinked and / or non-crosslinked polysaccharide, optionally an anesthetic agent, from the degradation of its rheological properties during sterilization, preferably by heat.

17. Use of a solution comprising zinc ions and citrate ions to preserve the stability over time of a hydrogel comprising a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof and optionally an anesthetic agent.