Hydrogels for soft tissue filling

Abstract

The present invention relates to hydrogels that can maintain their initial thickness over time. The projection index PIdx of the crosslinked polysaccharide-based hydrogel is greater than or equal to 80%.

Description

Technical Field

[0001] The present invention relates to filling gels, in particular filling gels based on cross-linked polysaccharides, for use in the aesthetic and / or medical fields for filling wrinkles and / or providing more relief to certain areas of the face. Background Art

[0002] The filling gel is, for example, a polysaccharide-based hydrogel, in particular hyaluronic acid (HA), and is injected under the skin using a syringe. Hydrogels suitable for filling soft tissues are usually cross-linked hydrogels, i.e., the polysaccharides are cross-linked by one or more cross-linking agents. This cross-linking enables the hydrogel to have the desired mechanical properties for filling soft tissues.

[0003] Manufacturers propose filling hydrogels with various rheological properties depending on the site of injection of the hydrogel. For example, in the case of injection into the outer layers of the skin, the hydrogel should lightly fill fine wrinkles and lines and be able to easily follow facial movements. However, hydrogels intended for mid- or deep-seated implantation, particularly hydrogels intended for filling more severe folds and wrinkles and / or for creating volume (referred to as "volumizer" hydrogels), must advantageously have the inherent ability to continuously maintain their thickness in the skin layers, including under the stress of facial movements.

[0004] The most commonly used rheological measurements of hydrogels are those of the G' and G" moduli, to which the phase shift angle δ is related (tan δ = G" / G'). These measurements are usually made in their linear viscoelastic region at low oscillatory stresses with low amplitudes. These measurements do not necessarily reflect the mechanical stresses and deformations to which the filled hydrogels are subjected in vivo. These measurements cannot predict the behavior of the hydrogel under compression. However, in the case of implantation (especially deep implantation), the hydrogel is compressed between tissue layers, and its ability to produce relief depends on its ability not to diffuse or excessively lose its thickness over time.

[0005] It is known that after in vivo application, commercial hydrogels known as "volumizers" are often ineffective in maintaining their overall thickness under the stress of surrounding tissues and / or facial movement. Therefore, it is of interest to develop volumizer hydrogels that can consistently maintain their thickness in the skin layers, including under the stress of facial movement.

[0006] Furthermore, crosslinked hydrogels may have biocompatibility issues. In order to optimize this biocompatibility, one solution consists in reducing the degree of modification of the polysaccharides required to obtain the gel, thereby making it closest to the natural polysaccharides, or choosing an alternative crosslinking method, such as crosslinking by sol-gel reaction. However, using such solutions, the hydrogels prepared do not have mechanical properties suitable for use as volumizers, in particular these hydrogels may be less crosslinked, may have a reduced G' modulus and a lower ability to maintain their thickness in tissue. Therefore, there is a need to provide hydrogels (particularly volumizer hydrogels) that have a minimal degree of polysaccharide modification, but have mechanical properties suitable for creating volume and sustainably maintaining their thickness in the skin layers (including under the stress of facial movements).

[0007] The present invention therefore aims to provide a hydrogel based on cross-linked polysaccharides having an improved volumizing capacity in vivo, in other words the ability to sustainably maintain its thickness in the skin layers, including under the stress of facial movements. The proposed hydrogel will advantageously be biocompatible and natural and will be as little modified as possible. Summary of the invention

[0008] The present invention relates to a hydrogel based on cross-linked polysaccharides, the projection index PIdx of the hydrogel being greater than or equal to 80%, the projection index being determined according to the method described in detail below.

[0009] The present invention also relates to a cross-linked polysaccharide-based hydrogel prepared by a method comprising the following steps, wherein the projection index PIdx of the hydrogel is greater than or equal to 80%:

[0010] a) providing at least one polysaccharide or a salt thereof;

[0011] b) providing at least one cross-linking agent or a salt thereof;

[0012] c) preparing a cross-linking reaction medium comprising one or more polysaccharides, one or more cross-linking agents and a solvent, wherein the total amount of the cross-linking agent ranges from 0.001 mol to 0.03 mol, preferably from 0.001 mol to 0.02 mol, per 1 mol of polysaccharide repeating unit, the duration of the reaction medium preparation step preferably not exceeding 5 hours; and

[0013] d) placing the reaction medium obtained at the end of step c) under a pressure P and a temperature T, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P, preferably for a duration ranging from 1 week to 17 weeks.

[0014] The present invention also relates to a cross-linked polysaccharide-based hydrogel prepared by a method comprising the following steps, wherein the projection index PIdx of the hydrogel is greater than or equal to 80%:

[0015] a) providing at least one polysaccharide or a salt thereof;

[0016] b) providing at least one functionalizing agent or a salt thereof;

[0017] c) preparing a cross-linking reaction medium comprising one or more polysaccharides, one or more functionalizing agents and a solvent, the total amount of the functionalizing agents being in the range of 0.001 mol to 0.50 mol, preferably 0.005 mol to 0.45 mol, in particular 0.10 mol to 0.25 mol, or in the range of 0.001 mol to 0.03 mol, preferably 0.001 mol to 0.02 mol, per 1 mol of polysaccharide repeating unit, the duration of the step of preparing the reaction medium preferably not exceeding 5 hours; and

[0018] d) placing the reaction medium obtained at the end of step c) under a pressure P and a temperature T, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P, preferably for a duration ranging from 1 week to 17 weeks.

[0019] The invention also relates to the use of the hydrogel of the invention for preventing and / or treating changes in the viscoelastic or biomechanical properties of the skin; for filling wrinkles, fine lines and scars; for reducing nasolabial folds and sadness lines; for increasing the volume of the cheekbones, chin or lips or for reducing the appearance of wrinkles and fine lines.

[0020] Other aspects of the invention are described in the claims and below.

[0021] definition

[0022] The term "gel" denotes a polymer network whose entire volume is swollen by a fluid. This means that the gel is formed by two media dispersed in each other, one being a "solid" and the other being a "liquid". The medium referred to as "solid" consists of long molecular polymers connected to each other by weak bonds (such as hydrogen bonds) or covalent bonds (crosslinking). The liquid medium consists of a solvent. The gel generally corresponds to a product having a phase angle δ at 1 Hz less than or equal to 45° when deformed by 0.1% or at a pressure of 1 Pa, advantageously a phase angle δ of 2° to 45° or 20° to 45°.

[0023] The term "hydrogel" means a gel as defined above in which the solvent constituting the liquid medium is predominantly water (e.g. at least 90% by weight of the liquid medium, in particular at least 95% by weight, in particular at least 97% by weight, in particular at least 98% by weight). Preferably, the liquid medium comprises, in particular consists of, a buffer solution which advantageously brings the pH of the liquid medium to between 6.8 and 7.8, in particular phosphate buffered saline.

[0024] The term "injectable gel" means a gel that can flow and can be manually injected through a syringe equipped with a needle with a diameter ranging from 0.1 mm to 0.5 mm (e.g., 32G, 30G, 27G, 26G, 25G hypodermic needle). Preferably, an "injectable gel" is a gel having an average extrusion force of less than or equal to 25 N, preferably in the range of 5 N to 25 N, and more preferably in the range of 8 N to 15 N, during measurement at ambient temperature using a dynamometer at a fixed speed of about 12.5 mm / min in a syringe with an outer diameter less than or equal to 0.4 mm (27G) and a needle with an outer diameter greater than or equal to 6.3 mm of 1 / 2 the length.

[0025] "Superficial application" means that the composition is superficially applied into or onto the skin, for example by mesotherapy, for treating the outer layer of the skin, the epidermis and the superficialmost part of the dermis, thereby reducing superficial wrinkles and / or improving the quality of the skin (e.g., its radiance, density or structure) and / or rejuvenating the skin.

[0026] "Mid dermis application" means applying the composition to the mid dermis portion of the skin, thereby treating the mid dermis of the skin and reducing mid dermis wrinkles.

[0027] "Deep application" means applying the hydrogel to the deepest layers of the skin, the subcutaneous tissue and the deepest part of the dermis and / or under the skin (above the periosteum) to "volumize" soft tissues, such as filling the deepest wrinkles and / or partially atrophic areas of the facial and / or body contours. So-called "volumizer" hydrogels can generally be applied for deep applications.

[0028] "Cross-linked polysaccharide" refers to a polysaccharide that is modified during a cross-linking reaction. The cross-linking can be performed by a cross-linking agent, or by a sol-gel reaction. When the cross-linking reaction is a sol-gel reaction, the polysaccharide is modified using a functionalizing agent.

[0029] In contrast, "uncrosslinked polysaccharide" refers to a polysaccharide that has not been modified using a crosslinking agent and / or a functionalizing agent and has therefore not undergone a crosslinking reaction.

[0030] The term "cross-linking agent" denotes any compound capable of initiating cross-links between different polysaccharide chains.

[0031] The term "functionalizing agent" means any compound capable of reacting with the functional groups of the polysaccharide and reacting via a sol-gel reaction.

[0032] The "molar crosslinking ratio" (TR) (expressed in %) represents the molar ratio of the amount of crosslinking agent to the amount of polysaccharide repeating units introduced into the crosslinking reaction medium (expressed relative to 100 moles of polysaccharide repeating units in the crosslinking medium).

[0033] The "molar functionalization rate" (expressed in %) represents the molar ratio of the amount of functionalizing agent to the amount of polysaccharide repeating units introduced into the crosslinking reaction medium (expressed relative to 100 moles of polysaccharide repeating units in the functionalizing medium).

[0034] The expression "repeating unit" of a polysaccharide refers to a structural unit composed of one or more (usually 1 or 2) monosaccharides, the repetition of which produces a complete polysaccharide chain.

[0035] The "degree of modification" (MOD) of a polysaccharide (e.g., hyaluronic acid) corresponds to the molar amount of cross-linking agent and / or functionalizing agent bound to the polysaccharide via one or more of its ends, expressed relative to 100 moles of polysaccharide repeating units. It can be determined by methods known to those skilled in the art, such as nuclear magnetic resonance spectroscopy (NMR) (see Experimental Section).

[0036] The term "polysaccharide" denotes a polymer composed of monosaccharides (preferably the D enantiomers) linked together by glycosidic bonds.

[0037] The term "ambient temperature" shall mean a temperature in the range of 20°C to 25°C, more particularly 25°C. DETAILED DESCRIPTION

[0038] The cross-linked polysaccharide-based hydrogels are characterized by a projection index PIdx greater than or equal to 80%, which is determined as described in detail below. The projection index reflects the rheological behavior of the hydrogel under force over time. A hydrogel with a projection index PIdx greater than or equal to 80% has the ability to maintain its initial thickness over time. In other words, such a hydrogel has the ability to maintain a thickness greater than or equal to 80% of its initial thickness over time. Therefore, they are particularly suitable as "volumizer hydrogels".

[0039] The Projection Index therefore makes it possible to differentiate between the hydrogels, retaining that one or those hydrogels having the most interesting properties for the desired result, in particular when seeking to obtain so-called "volumizer" hydrogels.

[0040] Determination of projection index

[0041] The projection index is determined by a method comprising the following steps:

[0042] 1) subjecting a one gram pellet of a hydrogel, previously placed between two circular pressure plates of a rheometer and having an initial thickness d0 of 700 μm given by the distance between the two plates, the two plates being parallel and having a diameter of 40 mm, to a fixed constant compressive force F of 2 Newtons,

[0043] 2) collecting the development of the thickness change of the hydrogel compressed in this manner at 25° C. for 1 hour;

[0044] 3) Parameterizing a generalized Maxwell model that approximates the development observed from the acquisition performed in step 2), the model representing the change in thickness of the hydrogel over time d gel (t), and corresponds to the following equation 1:

[0045]

[0046] where d ∞ is the limiting thickness obtained at equilibrium, A i is a constant, τi is the relaxation parameter, and the number of members i of the equation is equal to 2;

[0047] 4) Determine from the model the ultimate thickness d to which the hydrogel tends to develop over time ∞ ;as well as

[0048] 5) Generate the projection index PIdx by the following equation 2:

[0049]

[0050] The ultimate thickness obtained at equilibrium (d ∞ ) represents the thickness obtained under the applied compressive force F for an infinite time.

[0051] The applied compressive force F (2N) is preferably in the region of linear viscoelastic deformation of the hydrogel, in other words, the applied compressive force F advantageously behaves as a pressure in the linear viscoelastic region of the hydrogel. Such force values ​​are suitable for so-called volumizing gels. The linear viscoelastic region (LVER) corresponds to the range of hydrogel deformation from the initial value of the elastic modulus E' to a value where the elastic modulus E' decreases by 10% from its initial value.

[0052] Therefore, the method for determining the projection index may initially include a step of determining the linear viscoelastic region of the hydrogel. This step can ensure that the applied compressive force F is indeed in the region of linear viscoelastic deformation of the gel. Advantageously, this step includes performing an oscillatory stress sweep measurement in compression mode at a given oscillation frequency, thereby determining the linear viscoelastic region and setting limits for the applied normal force. The measurement is applied within a stress range determined under the same conditions as the measurement conditions of the projection index. Preferably, at 1 Hz and 25°C, the stress range covers 10 Pa to 10000 Pa, and the plate geometry is a parallel plate (anodized aluminum with a diameter of 40 mm, TA ), and the distance between the substrate and the pressure plate is equal to 700μm.

[0053] When the applied compressive force F is in the linear viscoelastic region LVER of the hydrogel, the projection index PIdx is a direct measure of the ability of the hydrogel to maintain its initial thickness under a given compressive stress. Therefore, the projection index reflects the behavior of the hydrogel within the tissue.

[0054] When the applied compressive force F exceeds its linear elastic region LVER, the projection index PIdx gives an indication of its ability to maintain its thickness for a certain time; in theory, in an unconstrained geometry, these hydrogels will continue to creep, but in practice the surrounding tissue will act as a certain limit, thereby preventing the gel from creeping, so PIdx is still a useful piece of information for predicting the behavior of the gel in vivo.

[0055] The projection index is preferably measured using a combination of TRIOS software (TA ) with parallel plate geometry (40 mm diameter, anodized aluminum, TA ) is carried out using a rheometer DHR2 equipped with a 200 μm flowmeter. More specifically, the determination of the projection index can be carried out in the following manner. A 1 gram hydrogel pellet (sample) is deposited on the substrate of the rheometer. A pressure plate with a diameter of 40 mm applies a force to the sample until its thickness (given by the distance between the substrate and the pressure plate) is equal to 700 μm (d0).

[0056] After the thickness reaches this predetermined value d0, preferably after the resistance of the hydrogel is less than or equal to the value of the force F to be applied, a constant fixed compressive force F of 2N (Newton) is applied and the development of the thickness variation of the hydrogel is collected. The collection is started by taking this moment as the time base point of the collection. This collection is carried out at 25°C for 1 hour. Preferably, the use of Software (version 9.6.0.172) calculates the parameter d ∞ .

[0057] The software is used to apply the mathematical model in such a way that there is an optimal adjustment of the model according to the observed thickness development.

[0058] The model used is the generalized Maxwell model, which gives the development of the hydrogel thickness as a function of time t by:

[0059]

[0060] Good results are obtained from the presence of two exponential terms:

[0061]

[0062] d ∞ corresponds to the thickness at equilibrium after infinite time, A i is a constant, τi is the relaxation parameter, and the number of members i of the equation is equal to 2.

[0063] The software is able to iteratively calculate / determine A i and the value of τi. i and τi reflect the physical relaxation of the hydrogel. They are positive. In particular, A i (corresponding to thickness) is greater than 0 and less than or equal to 700 μm. In particular, τi (corresponding to time) is less than 24 hours, preferably less than 2 hours, more preferably less than 1 hour. In particular, when R 2 When >0.99, stop iteration.

[0064] Then, the projection index value is calculated as follows:

[0065]

[0066] The value d0 corresponds to the initial thickness between the plates before the force is applied.

[0067] The fixed parameters applied for the method for determining the projection index and thus characterizing the hydrogels according to the invention enable easy differentiation of the projection index values ​​while applying a compressive force in the LVER region of the so-called "volumizer" hydrogel.

[0068] Hydrogel

[0069] The cross-linked polysaccharide-based hydrogel of the present invention has a projection index greater than or equal to 80%, preferably greater than or equal to 82%, more preferably greater than or equal to 85%, and more preferably greater than or equal to 90%. The projection index is determined by the method detailed above.

[0070] In certain embodiments, the projection index PIdx of the hydrogel according to the present invention is greater than or equal to 81%, preferably greater than or equal to 82%, more preferably greater than or equal to 83%, still more preferably greater than or equal to 84%.

[0071] In certain embodiments, the projection index PIdx of the hydrogel according to the present invention is greater than or equal to 86%, preferably greater than or equal to 87%, more preferably greater than or equal to 88%, still more preferably greater than or equal to 89%.

[0072] A hydrogel having a projection index greater than or equal to 80% has the ability to sustainably maintain its thickness in the skin layers, including under the stress of facial movements, compared to a hydrogel having a lower projection index. In particular, the hydrogel according to the invention is a volumizer hydrogel intended for filling severe folds and wrinkles and / or for creating volume.

[0073] The hydrogel according to the invention is a cross-linked polysaccharide-based hydrogel. Therefore, the hydrogel according to the invention comprises one or more cross-linked polysaccharides. Cross-linking enables the hydrogel to be obtained with the desired mechanical properties for filling soft tissue.

[0074] Polysaccharide can be any polymer or its mixture consisting of monosaccharide linked together by glycosidic bond.Preferably, polysaccharide is selected from pectin and pectin material, chitosan, chitin, cellulose and derivatives thereof, agarose, mucopolysaccharide (such as hyaluronic acid, heparin precursor, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate) and its mixture.Also more preferably, polysaccharide is selected from hyaluronic acid, heparin precursor, chondroitin and its mixture, and even more preferably, polysaccharide is one of hyaluronic acid or its salt, particularly physiologically acceptable salt, such as sodium salt, potassium salt, zinc salt, calcium salt, magnesium salt, silver salt, calcium salt and its mixture.More particularly, hyaluronic acid is the form (NaHA) of its acid form or sodium salt.Therefore, hydrogel can be the hydrogel based on one of hyaluronic acid and / or its salt.

[0075] The weight average molar mass of the polysaccharide generally ranges from 0.03 MDa to 10 MDa.

[0076] Preferably, if the polysaccharide is hyaluronic acid or one of its salts, its weight average molar mass (Mw) ranges from 0.05 MDa to 10 MDa, preferably from 0.5 MDa to 5 MDa, for example from 0.07 MDa to 10 MDa or from 0.07 MDa to 5 MDa, or from 0.5 MDa to 5 MDa or from 1 MDa to 5 MDa or from 2 MDa to 4 MDa.

[0077] Crosslinking agent

[0078] The polysaccharide can be cross-linked by a cross-linking agent, which is preferably selected from a bifunctional or multifunctional epoxy cross-linking agent or a non-epoxy cross-linking agent, in other words, prepared by the reaction of the polysaccharide with the cross-linking agent. The epoxy agent can include 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, 1,2-bis(2,3-epoxypropyl)-2,3-ethane (EGDGE), poly(ethylene glycol) diglycidyl ether (PEGDG) and mixtures thereof. The non-epoxy agent can include endogenous polyamines (e.g., spermine, spermidine and putrescine), aldehydes (e.g., glutaraldehyde), carbodiimide and divinyl sulfone, derivatives of hydrazide (e.g., adipic acid dihydrazide), bis-alkoxyamines, dithiols (e.g., polyethylene glycol dithiol) and mixtures thereof. Non-epoxidizing agents may include amino acids (eg, cysteine, lysine), peptides or proteins comprising amino acids (eg, cysteine, lysine), trimetaphosphates (eg, sodium trimetaphosphate, calcium trimetaphosphate, or even barium trimetaphosphate).

[0079] In certain embodiments, the crosslinking agent is an epoxidizing agent, which is preferably 1,4-butanediol diglycidyl ether (BDDE) or polyethylene glycol-diglycidyl ether. The crosslinking agent is preferably 1,4-butanediol diglycidyl ether (BDDE).

[0080] In certain embodiments, the crosslinking agent is a non-epoxy agent, preferably selected from endogenous polyamines, aldehydes, carbodiimides, divinyl sulfones, amino acids, peptides, and mixtures thereof.

[0081] The hydrogel according to the present invention is preferably a cross-linked polysaccharide-based hydrogel with a molar cross-linking rate of less than or equal to 3%. More preferably, the hydrogel according to the present invention is a cross-linked polysaccharide-based hydrogel with a molar cross-linking rate greater than 0% and less than or equal to 2%, preferably less than or equal to 1.5%, more preferably less than or equal to 1%, still more preferably less than or equal to 0.8% or 0.5%, in particular in the range of 0.1% to 0.8% or 0.1% to 0.5% (moles of cross-linking agent / 100 moles of repeating units of one or more polysaccharides).

[0082] Functionalizing agent

[0083] The polysaccharide can be cross-linked by a functionalizing agent. The functionalizing agent is capable of cross-linking the polysaccharide by a sol-gel reaction. Thus, the functionalizing agent generally comprises a single functional group capable of reacting with a functional group of the polysaccharide and comprises a silylated group capable of reacting with another silylated group by a sol-gel reaction, thereby being able to cross-link the polysaccharide and form a hydrogel.

[0084] The functionalizing agent is typically a molecule of Formula II as shown below, or a salt thereof:

[0085]

[0086] in:

[0087] T represents an isocyanate group, an amino group, an epoxide group, a carboxyl group, an N-succinimidyloxycarbonyl group, an N-sulfosuccinimidyloxycarbonyl group, a halocarbonyl group, an isothiocyanate group, a vinyl group, a formyl group, a hydroxyl group, a mercapto group, a hydrazino group, an acylhydrazino group, an aminooxy group, a carbodiimide group or an acid anhydride residue;

[0088] A represents a chemical bond or a spacer group;

[0089] R5 and R6 are the same or different and represent a hydrogen atom; a halogen atom; an -OR4 group, wherein R4 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms; an aryl group; or an aliphatic hydrocarbon group having 1 to 6 carbon atoms optionally substituted by one or more groups selected from halogen atoms, aryl groups and hydroxyl groups;

[0090] R10 represents a hydrogen atom, an aromatic group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms.

[0091] Preferably, in formula II, T represents an isocyanate group, a mercapto group, an amino group, an epoxide group, a vinyl group, a formyl group or a carbodiimide group, more advantageously, T represents an epoxide group or an amino group, still more advantageously, T represents an epoxide group.

[0092] Preferably, in formula II, A represents a spacer group, more preferably, in particular a linear or branched and saturated divalent aliphatic hydrocarbon chain having 1 to 12 carbon atoms:

[0093] - wherein one or more (especially 1, 2, 3 or 4) divalent units are optionally inserted between two carbon atoms of the chain, the divalent units being selected from arylene, -O-, -S-, -S(O)-, -C(=O)-, -SO2- and -N(R9)-, wherein R9 represents a hydrogen atom, an aliphatic hydrocarbon group having 1 to 6 carbon atoms, or an aryl-(C1-C6)alkyl group,

[0094] - said chain is unsubstituted or substituted by one or more monovalent groups selected from halogen atoms, hydroxyl groups, aryl-(C1-C6)alkyl groups.

[0095] Advantageously, A is an aliphatic divalent hydrocarbon chain, in particular a linear or branched and saturated aliphatic divalent hydrocarbon chain, in which one or more -O-divalent units, more advantageously 1 to 4 -O-divalent units, still more advantageously one divalent -O-unit are optionally inserted between two carbon atoms of the chain.

[0096] Preferably, A is a (C1-C12) alkylene chain, wherein one or more divalent -O- units, more preferably 1 to 4 divalent -O- units, still more preferably one divalent -O- unit are optionally inserted between two carbon atoms of the chain.

[0097] In particular, in the molecule of formula II, A represents a divalent -(C1-C6)alkylene-O-(C1-C6)alkylene-chain, in particular a -(C1-C4)alkylene-O-(C1-C4)alkylene chain, more particularly a divalent -CH2-O-(CH2)3-chain, the CH2 group being bonded to T and the (CH2)3 group being bonded to Si.

[0098] Advantageously, the spacer group will also be able to avoid steric hindrance between the silylated group and the T group of the molecule of formula II, thus ensuring a stable bond between these two groups.

[0099] Preferably, in Formula II, R5 and R6 are the same or different and represent an -OR4 group (wherein R4 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms) or an aliphatic hydrocarbon group having 1 to 6 carbon atoms optionally substituted by one or more groups selected from halogen atoms, aryl groups and hydroxyl groups.

[0100] In particular, R5 and R6 are identical or different and represent a -OR4 group (wherein R4 represents (C1-C6)alkyl) or a (C1-C6)alkyl group.

[0101] Advantageously, R5 and R6 are identical or different and represent a -OR4 group (wherein R4 represents a hydrogen atom, an aromatic group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms), preferably, R4 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms, for example a (C1-C6) alkyl group.

[0102] Preferably, in Chemical Formula II, R10 represents a hydrogen atom or an aliphatic hydrocarbon group having 1 to 6 carbon atoms (e.g., a (C1-C6) alkyl group), more advantageously, R10 represents an aliphatic hydrocarbon group having 1 to 6 carbon atoms, e.g., a (C1-C6) alkyl group.

[0103] Preferably, the molecule of formula II satisfies:

[0104] -T is as defined above and advantageously represents an amino group or an epoxide group, preferably an epoxide group;

[0105] - in the molecule of formula II, A is a divalent -(C1-C6)alkylene-O-(C1-C6)alkylene-chain, in particular -(C1-C4)alkylene-O-(C1-C4)alkylene-, for example -CH2-O-(CH2)3-, the CH2 group preferably being bonded to T and the (CH2)3 group being bonded to Si;

[0106] - R5 and R6 are identical or different and are each an -OR4 group (wherein R4 represents a (C1-C6)alkyl group, preferably a methyl or ethyl group) or a (C1-C6)alkyl group, preferably a methyl or ethyl group; and

[0107] - R10 is (C1-C6) alkyl, preferably methyl or ethyl;

[0108] The R5, R6 and OR10 groups may be the same.

[0109] In particular, the molecule of formula II is selected from (3-aminopropyl)triethoxysilane (APTES), (3-glycidyloxypropyl)trimethoxysilane (GPTMS), 3-glycidoxypropyldimethoxymethylsilane, 3-glycidoxypropyldimethylethoxysilane, (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane and the like. The mixture is preferably selected from (3-glycidyloxypropyl)trimethoxysilane (GPTMS), (3-glycidyloxypropyl)ethoxydimethoxysilane, (3-glycidyloxypropyl)triethoxysilane, diethoxy(3-glycidyloxypropyl)methylsilane and mixtures thereof, and more preferably (3-aminopropyl)triethoxysilane (APTES), (3-glycidyloxypropyl)trimethoxysilane (GPTMS) and mixtures thereof.

[0110] The hydrogel according to the present invention is preferably a cross-linked polysaccharide-based hydrogel, and its molar functionalization rate is less than 50%. More preferably, the hydrogel according to the present invention is a cross-linked polysaccharide-based hydrogel, and its molar functionalization rate ranges from 1% to 50% or 5% to 45% or 10% to 25% (number of moles of functionalizing agent / 100 moles of repeating units of one or more polysaccharides). In certain embodiments, the hydrogel according to the present invention is a cross-linked polysaccharide-based hydrogel, and its molar functionalization rate ranges from 0.1% to 3% or 0.1% to 2%.

[0111] The hydrogel according to the present invention is preferably a cross-linked polysaccharide-based hydrogel having a degree of modification (MOD) of less than or equal to 3%, preferably less than or equal to 2%, more preferably less than or equal to 1%.

[0112] Advantageously, the hydrogel according to the invention is a polysaccharide-based hydrogel having a degree of modification (MOD) less than or equal to 1.8%, preferably less than or equal to 1.5%, more preferably less than or equal to 1.2%, still more preferably less than 1%.

[0113] The degree of modification (MOD) / PIdx of the hydrogel according to the invention is advantageously less than or equal to 0.050, preferably less than or equal to 0.040, more preferably less than or equal to 0.030, more preferably less than or equal to 0.015.

[0114] Advantageously, the degree of modification (MOD) / PIdx of the hydrogel according to the invention is less than or equal to 0.038, preferably less than or equal to 0.025, more preferably less than or equal to 0.020 and even more preferably less than or equal to 0.012.

[0115] Relative to the gross weight of the hydrogel, the hydrogel according to the present invention can include 0.1 wt % to 5 wt %, preferably 1 wt % to 3 wt % of a polysaccharide (e.g., hyaluronic acid), which is present in a cross-linked form. The hydrogel can also include a polysaccharide in an uncross-linked form. In particular, relative to the gross weight of the polysaccharide (e.g., hyaluronic acid) present in the hydrogel, the content of the uncross-linked polysaccharide (e.g., hyaluronic acid) can be from 0.5 wt % to 40 wt %, preferably 1 wt % to 40 wt %, more preferably 2 wt % to 30 wt %, and even more preferably 2 wt % to 20 wt % changes.

[0116] The total concentration of polysaccharide in the hydrogel advantageously varies from 1 mg / g hydrogel to 50 mg / g hydrogel, more advantageously from 5 mg / g hydrogel to 35 mg / g hydrogel, and even more advantageously from 10 mg / g hydrogel to 30 mg / g hydrogel. This polysaccharide is preferably hyaluronic acid, and even more preferably sodium hyaluronate.

[0117] The hydrogel according to the present invention may comprise one or more polysaccharides, which are advantageously subjected to the following steps: crosslinking for 1 to 17 weeks or 2 to 17 weeks at a pressure P and a temperature T (the pressure P is less than or equal to atmospheric pressure, the temperature T is greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P).

[0118] The pressure P is preferably atmospheric pressure. "Atmospheric pressure" is the pressure exerted by the air that makes up the atmosphere on any surface in contact with it.

[0119] The temperature of the reaction medium (temperature T) is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably in the range of -35°C to -10°C. Still more preferably, temperature T is about -20°C.

[0120] The hydrogel according to the invention is preferably a sterile hydrogel, in particular sterilized by heating (generally at a plateau temperature of 121° C. to 135° C.), preferably with a plateau duration ranging from 1 minute to 20 minutes, with F0 ≥ 15. The sterilization value F0 corresponds to the time (in minutes) required to inactivate 90% of the number of microorganisms present in the product to be sterilized at 121° C. Alternatively, sterilization can be carried out by using gamma rays, UV radiation or by ethylene oxide.

[0121] The hydrogel has a physiological pH, ie in the range of 6.8 to 7.8. The pH of the hydrogel according to the invention 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.

[0122] The hydrogel according to the invention is preferably an injectable hydrogel, in other words it is flowable and can be manually injected by means of a syringe equipped with a needle having a diameter ranging from 0.1 mm to 0.5 mm, such as a 32G, 30G, 27G, 26G, 25G hypodermic needle.

[0123] Additional Ingredients

[0124] In certain embodiments, the hydrogel of the present invention further comprises an additional component selected from one of an anesthetic, an antioxidant, an amino acid, a peptide, a protein (e.g., elastin), a vitamin, a mineral, a nucleic acid, a nucleotide, a nucleoside, a coenzyme, an adrenaline derivative, monohydrate and / or dihydrate sodium dihydrogen phosphate, sodium chloride, and mixtures thereof.

[0125] Uncrosslinked polysaccharides, in particular uncrosslinked hyaluronic acid, uncrosslinked heparin precursor or mixtures thereof, are examples of lubricants.

[0126] Examples of anesthetic agents include, by way of non-limiting example, amocaine, isopentocaine, aprindine, atocaine, articaine, benzocaine, betocaine, bupivacaine, butacaine, butamben, butancaine, chlorobutanol, chloroprocaine, cinchocaine, chlordacaine, cocaine, cleflurane, cyclomethicaine, dexicaine, diaminocaine, dipedoporin, dyclonine, etidocaine, euprocaine, febuvirine, formocaine, guanifecanol, encapcaine, hexacane, hydroxyprocaine, hydroxytetracaine, butamben, leuprocaine, levobupivacaine, and succinic acid. , levoxadrol, lidamidine, lidocaine, lotocaine, menglilate, mapivocaine, mepracaine, myticaine, octacaine, octadorine, oxycaine, oxybuprocaine, p-ethoxycaine, pidocaine, horocaine, piperocaine, pidocaine, polidocanol, pramoxine, prilocaine, procaine, propancaine, propivacaine, propoxycaine, proparacaine, pyrocaine, quatacaine, quinicaine, lisocaine, rhodocaine, ropivacaine, tetracaine, tolicaine, mesocaine and salts thereof (particularly hydrochloride) or mixtures thereof. Preferably, the hydrogel according to the invention comprises an anesthetic as defined above, in particular lidocaine, carbocaine or one of its salts, such as the hydrochloride.

[0127] In certain embodiments, the hydrogel according to the invention comprises an anesthetic, in particular carbocaine, lidocaine or one of their salts, more in particular the hydrochloride, preferably in an amount ranging from 0.1 mg / ml to 30 mg / ml, such as from 0.5 mg / ml to 10 mg / ml or more preferably from 2 mg / ml to 5 mg / ml.

[0128] Examples of antioxidants include, by way of non-limiting example, glutathione, reduced glutathione, ellagic acid, spermine, resveratrol, retinol, L-carnitine, polyols, polyphenols, flavonols, theaflavins, catechins, caffeine, ubiquinol, ubiquinone, alpha-lipoic acid and derivatives thereof, and mixtures thereof.

[0129] Examples of amino acids include, in a non-limiting manner, 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, derivatives thereof (e.g., N-acetyl derivatives, such as N-acetyl-L-cysteine), and mixtures thereof.

[0130] Examples of vitamins and their salts include, in a non-limiting manner, vitamins E, A, C, B, in particular vitamins B6, B8, B4, B5, B9, B7, B12, preferably pyridoxine and its derivatives and / or salts, preferably pyridoxine hydrochloride.

[0131] Examples of minerals include, in a non-limiting manner, zinc salts (e.g., zinc acetate (particularly zinc acetate dehydrate), zinc citrate), magnesium salts, calcium salts (e.g., hydroxyapatite, particularly in bead form), potassium salts, manganese salts, sodium salts, copper salts (e.g., copper sulfate, particularly copper sulfate pentahydrate) (optionally in hydrated form), and mixtures thereof.

[0132] Examples of nucleic acids include, in a non-limiting manner, adenosine, cytidine, guanosine, thymidine, cytosine, derivatives thereof, and mixtures thereof. Coenzymes include coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

[0133] Adrenaline derivatives include epinephrine, norepinephrine, and mixtures of these.

[0134] Method for preparing hydrogel

[0135] The hydrogel according to the present invention can be obtained by any preparation method known to those skilled in the art.

[0136] In particular, the hydrogel can be prepared by a method comprising the following steps:

[0137] a) providing at least one polysaccharide or a salt thereof;

[0138] b) providing at least one cross-linking agent or a salt thereof;

[0139] c) preparing a cross-linking reaction medium comprising one or more polysaccharides, one or more cross-linking agents and a solvent, wherein the total amount of the cross-linking agent ranges from 0.001 mol to 0.03 mol per 1 mol of polysaccharide repeating unit, the duration of the reaction medium preparation step preferably not exceeding 5 hours; and

[0140] d) Cross-linking reaction: placing the reaction medium obtained at the end of step c) under conditions capable of cross-linking the polysaccharide by reaction of the polysaccharide with one or more cross-linking agents, i.e., under pressure P and temperature T, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P, preferably for a duration ranging from 1 week to 17 weeks or from 2 weeks to 17 weeks.

[0141] In a variant of the above method for preparing a hydrogel, the crosslinking agent is replaced by a functionalizing agent. In this variant, the crosslinking reaction d) is carried out by a sol-gel reaction of a functionalized polysaccharide obtained by reacting a polysaccharide with one or more functionalizing agents; the crosslinking reaction d) is carried out at a pressure P and a temperature T, the pressure P is less than or equal to atmospheric pressure, the temperature T is greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P, preferably for a duration ranging from 1 week to 17 weeks or from 2 weeks to 17 weeks. In this variant of the method for preparing a hydrogel, step b) comprises providing at least one functionalizing agent, step c) comprises preparing a reaction medium comprising one or more polysaccharides, one or more functionalizing agents and a solvent, the total amount of the functionalizing agent generally ranging from 0.001 mol to 0.50 mol per 1 mol of polysaccharide repeating unit.

[0142] The cross-linked polysaccharide-based hydrogels prepared by the above method are highly biocompatible. In fact, such hydrogels are prepared with a small amount of cross-linking agent. However, they have mechanical properties suitable for generating volume.

[0143] Furthermore, the hydrogel prepared by this method and having a projection index PIdx greater than or equal to 80% can sustainably maintain its thickness in the skin layer (including under the stress of facial movements). This hydrogel is particularly suitable as a volumizing agent hydrogel.

[0144] Providing at least one polysaccharide or a salt thereof (step a))

[0145] Step a) of the process according to the invention for preparing a hydrogel comprises providing at least one polysaccharide or a salt thereof, in particular a physiologically acceptable salt.

[0146] The polysaccharide is as described above. Preferably, the polysaccharide is hyaluronic acid or a hyaluronate, preferably a sodium salt.

[0147] The polysaccharide can be provided in a completely or partially hydrated form or in a dry form, such as in the form of a powder or fiber. More particularly, in step a), the polysaccharide is provided in a dry form (e.g., in the form of a powder or fiber). When the polysaccharide is provided in a hydrated form, it is in the form of an uncrosslinked gel or solution. In particular, when the polysaccharide is in a hydrated form, it is an uncrosslinked aqueous gel or aqueous solution. More particularly, the polysaccharide is mixed with water, an optional added phosphate buffer or a supplementary phosphate buffer, in other words, may contain additional ingredients as defined above. Therefore, it should be understood that the uncrosslinked aqueous gel or aqueous solution of the polysaccharide does not contain sodium hydroxide.

[0148] Providing at least one cross-linking agent or a salt thereof and / or a functionalizing agent (step b))

[0149] Step b) of the method for preparing a hydrogel according to the invention comprises providing at least one cross-linking agent or a salt thereof and / or at least one functionalizing agent capable of cross-linking polysaccharides by a sol-gel reaction.

[0150] The crosslinking agent and the functionalizing agent are as described above.

[0151] Preparation of the cross-linking reaction medium (step c))

[0152] Step c) of the method for preparing a hydrogel according to the present invention comprises preparing a crosslinking reaction medium. The reaction medium comprises one or more polysaccharides, one or more crosslinking agents and / or functionalizing agents and a solvent.

[0153] 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% by weight, or at least 98% by weight of water relative to the total weight of the solvent). For example, an organic solvent (e.g., an alcohol (particularly ethanol) or DMSO) may be used to solubilize the crosslinker prior to addition to the aqueous reaction medium, for example when it involves poly(dimethylsiloxane) (CAS No. 130167-23-6) terminated with a diglycidyl ether at each end.

[0154] The reaction medium may also contain salts, pH adjusters (e.g. Bronsted bases, more preferably hydroxide salts, such as sodium hydroxide or potassium hydroxide), additional ingredients as described above, and mixtures thereof. The addition of Bronsted bases may be particularly necessary when the functional groups of the crosslinking agent have epoxide groups or vinyl groups. 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 Bronsted bases (e.g. sodium hydroxide) to the reaction medium, typically at a concentration between 0.10 M and 0.30 M.

[0155] The total amount of cross-linking agent in the reaction medium is less than or equal to 0.03 mole or less than or equal to 0.02 mole per 1 mole of polysaccharide repeating unit, preferably less than or equal to 0.015 mole, and more preferably less than or equal to 0.01 mole. It is preferably from 0.001 mole to 0.03 mole per 1 mole of polysaccharide repeating unit, preferably 0.001 mole to 0.02 mole or 0.001 mole to 0.015 mole per 1 mole of polysaccharide repeating unit, more preferably 0.001 mole to 0.01 mole per 1 mole of polysaccharide repeating unit, and more preferably 0.001 mole to 0.008 mole or 0.001 mole to 0.005 mole per 1 mole of polysaccharide repeating unit. When the polysaccharide is a mucopolysaccharide (e.g., hyaluronic acid), the repeating unit is a disaccharide unit.

[0156] The total amount of the functionalizing agent in the reaction medium generally varies from 0.001 mol to 0.50 mol, preferably 0.005 mol to 0.45 mol, in particular 0.10 mol to 0.25 mol per 1 mol of polysaccharide repeating unit, or from 0.001 mol to 0.03 mol, preferably 0.001 mol to 0.02 mol per 1 mol of polysaccharide repeating unit. For example, the total amount of the compound of formula II or its salt in the reaction medium generally varies from 0.001 mol to 0.50 mol, preferably 0.005 mol to 0.45 mol, in particular 0.10 mol to 0.25 mol per 1 mol of polysaccharide repeating unit, or from 0.001 mol to 0.03 mol, preferably 0.001 mol to 0.02 mol per 1 mol of polysaccharide repeating unit. In general, the higher the weight average molar mass Mw of the polysaccharide, the lower the functionalization rate will be in order to obtain a hydrogel with equivalent mechanical properties, in particular equivalent viscoelastic properties (in particular stress and / or phase angle δ at the intersection of the elastic moduli G', G' and G "). In other words, the higher the weight average molar mass Mw of the polysaccharide, the smaller the total amount of functionalizing agent (e.g., molecule of formula II) in the reaction medium will be. The functionalization of the polysaccharide is generally carried out in an aqueous reaction medium.

[0157] The mass concentration of the polysaccharide or polysaccharide salt in the reaction medium advantageously varies from 50 to 300 mg / g of solvent, preferably from 100 to 200 mg / g.

[0158] Step c) of the process generally comprises a step of homogenizing the reaction medium. The homogenization is generally carried out by three-dimensional stirring, stirring with a mixer, stirring with blades or stirring with a spatula.

[0159] The reaction medium is usually prepared from one or more polysaccharides in dry form. When the reaction medium is prepared from one or more polysaccharides in hydrated form, the uncrosslinked aqueous gel or aqueous polysaccharide solution used to prepare the reaction medium does not contain sodium hydroxide.

[0160] Step c) is generally carried out at a temperature ranging from 4°C to 35°C, preferably from 15°C to 25°C. Preferably, the duration of step c) does not exceed 5 hours. It generally varies from 15 minutes to 4 hours, preferably from 30 minutes to 2 hours. Therefore, before starting step d), the contact time of the polysaccharide (whether the polysaccharide is provided in dry form or in hydrated form) with the sodium hydroxide generally does not exceed 5 hours, for example from 15 minutes to 4 hours or from 30 minutes to 2 hours. The reaction medium obtained at the end of step c) is advantageously placed directly under the conditions of step d) according to the invention.

[0161] Crosslinking at temperature T and pressure P (step d)

[0162] The cross-linking step d) of the above method comprises cross-linking one or more polysaccharides. It comprises reacting or causing a reaction medium to react so as to obtain a cross-linked polysaccharide-based hydrogel.

[0163] Crosslinking can be performed by reaction of one or more polysaccharides with a crosslinking agent, or by sol-gel reaction of polysaccharides functionalized by reaction of the polysaccharides with a functionalizing agent.

[0164] The crosslinking is carried out by placing the reaction medium obtained at the end of step c) under conditions that allow the polysaccharide to be crosslinked (i.e. under a pressure P less than or equal to atmospheric pressure and a temperature T greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P), preferably for a duration ranging from 1 week to 17 weeks or from 2 weeks to 17 weeks.

[0165] The pressure P is advantageously equal to atmospheric pressure or less than atmospheric pressure.

[0166] The temperature of the freezing point of the reaction medium represents the temperature at which the mixture of the components of the reaction medium solidifies (in other words, becomes non-fluid) on a macroscopic scale. Below the freezing point, the mixture is in a frozen state characterized by the coexistence of components in solid form and in liquid form. The frozen state is maintained to the temperature of the eutectic point of the reaction medium.

[0167] The temperature of the eutectic point of the reaction medium represents the temperature below which the mixture of the components of the reaction medium changes from a frozen state (liquid and solid phases coexist) to a completely solid state (in other words, a state in which all components of the mixture are in solid form). The freezing point and eutectic point of a mixture depend on the pressure to which the mixture is subjected, and are therefore measured at pressure P.

[0168] The freezing point and the eutectic point can be determined by differential scanning calorimetry. This method allows the determination of phase transitions. For this purpose, the product to be investigated is gradually cooled until its phase transition is observed.

[0169] The temperature T is preferably greater than or equal to -55°C and less than or equal to -5°C, preferably in the range of -35°C to -10°C. Still more preferably, the temperature T is about -20°C.

[0170] The pressure P is preferably atmospheric pressure. "Atmospheric pressure" is the pressure exerted by the air that constitutes the atmosphere on any surface in contact with it. It varies with altitude. At an altitude of 0m, the average air pressure is 101325Pa. 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 about -20°C.

[0171] The crosslinking of the polysaccharide according to step d) can be carried out under vacuum, in particular at a pressure P less than atmospheric pressure, preferably at 0.7×10 5 Pa and 0.9×10 5 Pa (between 0.7 bar and 0.9 bar), preferably between 0.7×10 5 Pa and 0.8×10 5 Pa (between 0.7 bar and 0.8 bar).

[0172] The crosslinking step d) is generally carried out for a period of at least 1 hour, preferably at least 3 hours, preferably at least 72 hours, preferably up to 27 weeks. Preferably, the crosslinking step d) is carried out at temperature T and pressure P for a period of 1 week to 25 weeks or 1 week to 20 weeks or 1 week to 17 weeks or 2 weeks to 25 weeks, preferably 2 weeks to 20 weeks or 2 weeks to 17 weeks, still more preferably 3 weeks to 8 weeks or 4 weeks to 7 weeks.

[0173] Cross-linking by cross-linking agent

[0174] The crosslinking of the polysaccharides occurs mainly in step d) (although it can start from step c)). This step thus enables the polysaccharide chains to be crosslinked to each other. The functional groups of the crosslinking agent react with the functional groups present on the polysaccharides so as to bind the polysaccharide chains to each other and crosslink them by forming intermolecular bonds. The crosslinking agent can also react with the functional groups present on the same polysaccharide molecule to form intramolecular bonds. In particular, the functional groups of the crosslinking agent react with -OH or -COOH or even -CHO groups present on the polysaccharide (e.g. hyaluronic acid). A crosslinked polysaccharide is thus obtained comprising at least one crosslinking bond between two polysaccharide chains, the crosslinking bond being the residue of the crosslinking agent.

[0175] Crosslinking can be carried out in the presence of a variety of crosslinking agents. When crosslinking is carried out in the presence of a variety of crosslinking agents, the crosslinking agent can be added to the reaction medium simultaneously or at a separate time. Therefore, step d) can include repeated crosslinking steps. Therefore, crosslinking is carried out in the presence of a total amount of a crosslinking agent of 0.1 to 3 moles, or 0.1 to 2 moles, or 0.1 to 1.5 moles, or 0.1 to 1 mole, or 0.1 to 0.8 moles, or 0.1 to 0.5 moles of crosslinking agent (or its salt) per 100 moles of polysaccharide repeating units. The crosslinking conditions used (particularly the percentage of crosslinking agent, duration and temperature and the weight average molar mass (Mw) of the polysaccharide) are interdependent.

[0176] The lower the percentage of crosslinking agent, the longer the duration of the reaction must be in order to obtain equivalent mechanical properties of the resulting gel. In other words, the smaller the molar percentage of the crosslinking agent, the fewer reactive functional groups will be in the reaction medium, and the lower the probability of two groups meeting and reacting together, so in order for the functional groups to react with each other and form crosslinks, thereby obtaining a hydrogel with desired properties, the duration of the reaction must be longer.

[0177] In a variant of the method for preparing a hydrogel, this step d) may be performed at ambient temperature (20° C. to 25° C.) and for a duration shorter than the above duration (eg a duration ranging from 1 hour to 7 days).

[0178] Sol-gel cross-linking

[0179] Functionalization of polysaccharides

[0180] When the crosslinking is carried out by a sol-gel reaction, the crosslinking reaction d) is carried out by a sol-gel reaction of a functionalized polysaccharide obtained by reaction of the polysaccharide with one or more functionalizing agents. The functionalization of the polysaccharide and the sol-gel reaction can be continuous or at least partially simultaneous.

[0181] The polysaccharide is generally functionalized using at least one molecule of formula II as described above, in such a way that it becomes a carrier of Si-OR groups that will be able to react together and give rise to a cross-linked polysaccharide.

[0182] Since the molecule of formula II contains a single reactive functional group relative to the polysaccharide and can only be crosslinked by sol-gel reaction, it does not have the toxicity of conventional crosslinking agents: the molecule of formula II cannot be directly crosslinked with biological molecules (proteins, DNA, etc.). More specifically, the functional group T of the molecule of formula II reacts with the functional group present on the polysaccharide, thereby functionalizing the polysaccharide chain. In particular, therefore, the functional group T of formula II reacts with the -OH or -COOH group or even the CHO functional group present on the polysaccharide (e.g., hyaluronic acid). Therefore, a functionalized polysaccharide is obtained, the functionalized polysaccharide comprising a dangling bond on the polysaccharide chain, the dangling bond comprising an -A-Si(R5)(R6)OR10 group, the -A-Si(R5)(R6)OR10 group being derived from the molecule of formula II, the molecule being able to impart biological properties to the hydrogel.

[0183] The solvent is typically water, or a mixture comprising water and an organic solvent (eg an alcohol (especially ethanol) or DMSO; typically a mixture comprising at least 90 wt % water, or at least 95 wt % or at least 98 wt % water relative to the total weight of the solvent).

[0184] The reaction medium generally contains 0.001 to 0.50 moles, preferably 0.005 to 0.45 moles, especially 0.10 to 0.25 moles of the molecule of formula II or its salt per 1 mole of polysaccharide repeating unit, or 0.001 to 0.03 moles, preferably 0.001 to 0.02 moles of the molecule of formula II or its salt per 1 mole of polysaccharide repeating unit.

[0185] The mass concentration of polysaccharide in the reaction medium is advantageously between 50 mg / g of solvent and 300 mg / g of solvent, preferably between 100 mg / g of solvent and 200 mg / g of solvent.

[0186] In certain embodiments, in particular when T is an epoxide, the functionalization is carried out at a pH greater than or equal to 9, or greater than or equal to 10, more advantageously greater than or equal to 12, and in particular less than 14, for example less than or equal to 13.5. For this purpose, the reaction medium preferably comprises a Bronsted base, more preferably a hydroxide, still more preferably sodium hydroxide or potassium hydroxide. Advantageously, the reaction medium comprises sodium hydroxide or potassium hydroxide in a concentration between 0.10M and 0.30M.

[0187] In certain embodiments, in particular when T is an amino group, the functionalization is carried out at a pH less than 7, more advantageously greater than or equal to 4.5 and less than 7 or less than or equal to 6.5. For this purpose, the reaction medium preferably comprises a Bronsted acid, more preferably hydrochloric acid, sulfuric acid or acetic acid.

[0188] In certain embodiments, the functionalization of the polysaccharide is carried out at atmospheric pressure and at a temperature between 4° C. and 60° C., preferably between 10° C. and 50° C., more preferably between 15° C. and 25° C. In these embodiments, the duration of the functionalization reaction may vary from 1 hour to 2 weeks, more particularly 3 hours to 1 week, still more particularly 3 hours to 96 hours, e.g. 3 hours to 80 hours, in particular 3 hours to 75 hours.

[0189] In certain embodiments, in particular when functionalization and crosslinking of the polysaccharide occur simultaneously or partially simultaneously, the functionalization of the polysaccharide can be at least partially carried out at a temperature T greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P. Here, the pressure P is advantageously atmospheric pressure.

[0190] The higher the functionalization temperature, the shorter the functionalization time will be in order to obtain the same degree of modification by functionalization.

[0191] Sol-Gel Reaction

[0192] The "sol-gel reaction" involves the formation of Si-O-Si bonds from Si-OR groups, where R represents a hydrogen atom, an aromatic group, or an aliphatic hydrocarbon group having 1 to 6 carbon atoms. The reaction is carried out as follows:

[0193] (i) if R is not a hydrogen atom, performing a hydrolysis step of at least a portion of the Si-OR groups to produce Si-OH groups; and then

[0194] (ii) A step of condensing Si-OH groups two by two or condensing Si-OH groups and Si-OR groups to form Si-O-Si bonds.

[0195] The functionalized polysaccharides were cross-linked via a sol-gel reaction to produce a hydrogel.

[0196] When the polysaccharide chains are functionalized with molecules of formula II, this step enables the polysaccharide chains to be cross-linked to each other. More specifically, in this step, at least a portion of the Si-OR10 groups and optionally at least a portion of the SiOR4 groups will react in pairs, optionally after hydrolysis of these groups, to form Si-O-Si bonds. This means that two molecules of formula II grafted onto the polysaccharide chain will react together through their Si-OR10 (or SiOR4, if applicable) end groups and covalently bond by forming Si-O-Si bonds, thereby binding the polysaccharide chains together and cross-linking them.

[0197] In this way, a cross-linked polysaccharide is obtained which comprises cross-links between two polysaccharide chains, said cross-links comprising divalent -Si-O-Si- groups.

[0198] The pressure P is advantageously less than or equal to atmospheric pressure.

[0199] The crosslinking of the polysaccharide according to step d) can be carried out under vacuum, in particular at a pressure P less than atmospheric pressure, preferably at 0.7×10 5 Pa and 0.9×10 5 Pa (between 0.7 bar and 0.9 bar), preferably between 0.7×10 5 Pa and 0.8×10 5 Pa (between 0.7 bar and 0.8 bar).

[0200] The pressure P is advantageously atmospheric pressure.

[0201] In certain embodiments, the polysaccharide can be crosslinked by a crosslinking agent or by a sol-gel reaction (via a functionalizing agent). The reaction can occur sequentially or simultaneously.

[0202] Additional Steps

[0203] The method for preparing a hydrogel according to the invention may comprise one or more additional steps, such as steps of adding one or more additional ingredients, purification, sterilization, sieving, swelling and / or packaging.

[0204] The method for preparing a hydrogel according to the present invention may include the step of adding at least one additional ingredient. The additional ingredient may be selected from lubricants, cosmetic active ingredients (such as antioxidants, coenzymes, amino acids, vitamins, minerals and nucleic acids), therapeutic active ingredients (such as anesthetics, antibiotics, antifungal agents and adrenaline and its derivatives) and mixtures thereof. The additional ingredient may be as described above.

[0205] The method for preparing a hydrogel according to the invention may comprise at least one purification step. The purification may be performed by dialysis.

[0206] The method for preparing a hydrogel according to the invention may comprise a step of sterilizing the hydrogel. Sterilization is preferably carried out by heating. Sterilization is generally carried out by raising the temperature of the sterilization medium to a so-called "plateau temperature", maintaining the so-called "plateau duration" determined by said plateau temperature. Sterilization is preferably carried out at a plateau temperature in the range of 121° C. to 135° C., preferably with a plateau duration in the range of 1 minute to 20 minutes, wherein F0 ≥ 15. The sterilization value F0 corresponds to the time (in minutes) required to inactivate 90% of the number of microorganisms present in the product to be sterilized at 121° C. Alternatively, sterilization can be carried out in particular by using gamma rays, UV radiation or by ethylene oxide.

[0207] The method may comprise a step of sieving the hydrogel, more particularly using a sieve with a porosity between 50 μm and 2000 μm. This sieving step enables a more uniform hydrogel to be obtained with the most constant extrusion force (i.e. the most regular extrusion force). A person skilled in the art knows to select a sieve with an appropriate pore size according to the mechanical properties of the hydrogel being prepared.

[0208] The method may include a step of swelling the hydrogel. In the step of swelling the hydrogel, the concentration of the polysaccharide in the hydrogel is adjusted. In particular, a solvent is added, such as water, phosphate buffer, water for injection formulations. More particularly, the pH of the added solvent is about physiological pH (6.8-7.8). The concentration of the polysaccharide obtained in the hydrogel after the swelling step is advantageously varied from 1 mg / g gel to 50 mg / g hydrogel, more advantageously 5 mg / g hydrogel to 35 mg / g hydrogel, and even more advantageously 10 mg / g hydrogel to 30 mg / g hydrogel.

[0209] The method may include the step of packaging the hydrogel, for example in an injection device.

[0210] Where applicable, the step of adding one or more additional ingredients is preferably performed after the purification step.

[0211] Where applicable, the step of adding one or more additional ingredients is preferably performed prior to the sterilization step.

[0212] In particular, the step of adding one or more additional ingredients may also include adding at least one therapeutic active ingredient or at least one cosmetic active ingredient or a mixture thereof. When adding at least one therapeutic active ingredient and / or at least one cosmetic active ingredient, the step of adding one or more additional ingredients is preferably performed after step d).

[0213] Where applicable, a purification step is preferably performed after a step of adding one or more additional ingredients.

[0214] Where applicable, a purification step is preferably performed prior to a sterilization step.

[0215] Where applicable, a purification step is preferably performed prior to the sieving step.

[0216] The sterilization step is preferably performed after steps a) to d) and optional additional steps. In particular, the hydrogel is sterilized after packaging in its injection device, and packaging of the hydrogel is performed after all steps of the method and before sterilization.

[0217] application

[0218] The hydrogel according to the invention can be injected using any method known to the person skilled in the art. In particular, the hydrogel according to the invention can be administered by an injection device suitable for intraepidermal injection and / or intradermal injection and / or subcutaneous injection and / or periosteal injection. The injection device can be particularly selected from a syringe, a set of microinjectors, a wire, a laser or hydraulic device, an injection gun, a needle-free injection device or a microneedle roller.

[0219] The invention also relates to an injection device comprising the hydrogel according to the invention.

[0220] The hydrogel according to the invention is preferably injected subcutaneously.

[0221] The hydrogels according to the invention may be particularly suitable for deep applications.

[0222] The hydrogels according to the invention may have therapeutic and / or cosmetic applications.

[0223] In certain embodiments, the present invention also relates to a hydrogel for non-therapeutic use for filling and / or replacing tissue, particularly soft tissue, particularly by injecting the hydrogel into the tissue.

[0224] In the cosmetic field, the hydrogels according to the invention can be used in particular to compensate for the volume loss of tissues due to aging.

[0225] The hydrogel according to the invention can be used for preventing and / or cosmetically treating changes in the surface appearance of the skin. For example, the hydrogel according to the invention can be used in the cosmetic field for preventing and / or treating changes in the viscoelastic or biomechanical properties of the skin; for filling volume defects of the skin, in particular for filling wrinkles, fine lines and scars; for fading nasolabial folds and lines of sadness; for increasing the volume of the cheekbones, chin or lips; for restoring facial volume (in particular the cheeks, temples, facial contours and periocular area); for reducing the appearance of wrinkles and fine lines.

[0226] The invention also relates to the cosmetic use of a hydrogel as described above for filling tissue, in particular soft tissue, in particular to compensate for the loss of volume of tissue due to aging.

[0227] Example

[0228] 1. Equipment and Materials

[0229] -3MDa uncross-linked sodium hyaluronate,

[0230] -BDDE,

[0231] -0.25M NaOH,

[0232] -1M HCl,

[0233] - phosphate buffer,

[0234] -Lidocaine hydrochloride,

[0235] - 3D mixer,

[0236] -Rheometer DHR-2-TA

[0237] - Dynamometers and test benches,

[0238] - Paddle type crusher homogenizer,

[0239] - Polyethylene sterile bags.

[0240] 2. Methods

[0241] Comparison of the hydrogel according to the present invention (Gel A) with the commercially available volumizing hydrogel Juvederm -20mg / g RHA 4-23mg / g Volume-26mg / g(Merz ). The four hydrogels compared were sterile.

[0242] Unless otherwise stated, the steps described below were performed at ambient temperature (21°C).

[0243] Preparation of the hydrogel according to the invention :

[0244] BDDE and sodium hyaluronate (3 MDa, 120 mg / g) were dissolved in an aqueous sodium hydroxide solution to 0.25 M in a sterile bag. The mixture was then homogenized in a paddle mill until the sodium hyaluronate was completely dissolved (i.e. until a transparent and completely homogeneous product (i.e., the absence of any agglomerates) was obtained). The obtained mixture was kept at atmospheric pressure at -20°C for the duration indicated in Table 1. The pH of the obtained mixture was about 13.

[0245] Then, 1N HCl solution was added to the sterile bag until a pH of 7.3 ± 0.5 was obtained. The obtained mixture was diluted to a concentration of 25 mg hyaluronic acid per gram of gel A product using phosphate buffer solution PBS. The obtained mixture was homogenized for 24 hours by a three-dimensional stirrer. The mixture was dialyzed.

[0246] Then, a solution of sodium hyaluronate in an uncrosslinked form having a high molecular weight is added as a lubricant.

[0247] An aqueous solution of lidocaine hydrochloride was added to obtain 0.3% by weight of lidocaine hydrochloride relative to the weight of the product obtained.

[0248] The product thus obtained is sieved to micron size and then packaged in syringes.

[0249] Finally, the product was sterilized using an autoclave (plateau temperature between 121°C and 135°C with F0≥15).

[0250] The cross-linking conditions of the hydrogel according to the present invention are shown in Table 1 below.

[0251]

[0252] Table 1: Cross-linking conditions of hydrogels

[0253] The 2 Newton force applied in the method for determining the projection index was included in the LVER interval of the four hydrogels to be evaluated. The 3D images were taken at 25°C using a 3D imager equipped with TRIOS software (TA ) parallel plate geometry (40 mm diameter, anodized aluminum, TA The projection index values ​​of the four hydrogels were measured using a rheometer DHR2 with a 200 μm flowmeter. 1 gram of the hydrogel was applied between the plates, and the spacing between the plates corresponded to an initial thickness of 700 μm. Then, a compressive force of 2 Newtons was applied for 1 hour, and the development of the thickness variation of the hydrogel was collected. 2019 software (version 9.6.0.172) calculates the parameter d ∞ .

[0254] The generalized Maxwell model was applied, which gives the development of the hydrogel thickness as a function of time t by:

[0255]

[0256] where d ∞ is the limiting thickness obtained at equilibrium, A i is a constant, τi is the relaxation parameter, and the number of members i of the equation is equal to 2.

[0257] The results were obtained in the presence of two exponential terms:

[0258]

[0259] Then, the projection index value is calculated as follows:

[0260]

[0261] d0 is the initial thickness (700 μm), and d ∞ Obtained by applying the generalized Maxwell model.

[0262] The MOD of each hydrogel was determined by 1 H NMR measurement. The hydrogel was precipitated in isopropanol and dried under vacuum for 6 hours. The obtained hyaluronic acid residue was dissolved to 10 mg / mL in D2O. 50 μL of hyaluronidase (type VI-S from bovine testicles, 3 kU / mL in D2O) was added to degrade the gel at 37°C for 18 hours. The analysis was performed on a 400 MHz Bruker Avance spectrometer. The MOD was determined according to the following equation:

[0263] MOD(%)=(I δH1.6-1.7 / 4) / (I δH2.0-2.1 / 3)·100.

[0264] The minimum limit of quantification for MOD was 1%.

[0265] The results are shown in Table 2 below.

[0266]

[0267] Table 2: Results

[0268] The projection index PIdx of the hydrogel according to the present invention (Gel A) was greater than 80%, demonstrating a strong ability to sustainably maintain its thickness in the skin layer.

[0269] The projection index of the commercial hydrogel is less than 80%, which is less able to maintain its thickness under a force of 2 N than the hydrogel according to the present invention. In addition, the MOD / PIdx calculated for gel A is very small compared to the commercial hydrogel, demonstrating a stronger ability to sustainably maintain its thickness under a stress of 2 N while having a low degree of modification.

Claims

1. A cross-linked polysaccharide-based hydrogel, wherein the projection index PIdx of the cross-linked polysaccharide-based hydrogel is greater than or equal to 80%, and the projection index is determined according to a method comprising the following steps: 1) subjecting a one gram pellet of a hydrogel, previously placed between two circular pressure plates of a rheometer and having an initial thickness d0 of 700 μm given by the distance between the two plates, the two plates being parallel and having a diameter of 40 mm, to a fixed constant compressive force F of 2 Newtons, 2) collecting the development of the thickness change of the hydrogel compressed in this manner at 25° C. for 1 hour; 3) Parameterizing a generalized Maxwell model that approximates the development observed from the acquisition performed in step 2), the model representing the change in thickness of the hydrogel over time d gel (t), and corresponds to the following equation 1: where d ∞ is the limiting thickness obtained at equilibrium, A i is a constant, τi is the relaxation parameter, the number of members i of the equation is equal to 2, 4) Determine from the model the ultimate thickness d to which the hydrogel tends to develop over time ∞ ;as well as 5) Generate the projection index PIdx by the following equation 2:

2. The hydrogel according to claim 1, wherein The hydrogel has a ratio of degree of modification (MOD) / PIdx less than or equal to 0.050, preferably less than or equal to 0.040, more preferably less than or equal to 0.030, and even more preferably less than or equal to 0.

015.

3. The hydrogel according to claim 1 or 2, wherein the projection index PIdx of the hydrogel is greater than or equal to 82%, preferably greater than or equal to 85%, and more preferably greater than or equal to 90%.

4. A hydrogel according to any one of the preceding claims, wherein The degree of modification (MOD) of the polysaccharide is less than or equal to 3%, preferably less than or equal to 2%, more preferably less than or equal to 1%.

5. The hydrogel according to any one of the preceding claims, further comprising an uncrosslinked polysaccharide, preferably an uncrosslinked hyaluronic acid, an uncrosslinked heparin precursor or a mixture thereof.

6. A hydrogel according to any one of the preceding claims, wherein The cross-linked polysaccharide accounts for 0.1 to 5 wt %, preferably 1 to 3 wt %, relative to the total weight of the hydrogel.

7. The hydrogel according to any one of claims 1 to 6, wherein the total polysaccharide concentration in the hydrogel varies from 1 mg / g hydrogel to 50 mg / g hydrogel, preferably from 5 mg / g hydrogel to 35 mg / g hydrogel, and more preferably from 10 mg / g hydrogel to 30 mg / g hydrogel.

8. A hydrogel according to any one of the preceding claims, wherein The cross-linked polysaccharide is prepared by reacting a polysaccharide with a cross-linking agent, wherein the cross-linking agent is selected from 1,4-butanediol diglycidyl ether (BDDE), 1,2,7,8-diepoxy-octane, poly(ethylene glycol) diglycidyl ether (PEGDG), 1,2-bis(2,3-epoxypropoxy)ethane (EGDGE) and a mixture thereof.

9. The hydrogel according to claims 1 to 7, wherein The cross-linked polysaccharide is prepared by a sol-gel reaction of a polysaccharide functionalized with a functionalizing agent of formula II or a salt thereof in: -T represents an isocyanate group, an amino group, an epoxide group, a carboxyl group, an N-succinimidyloxycarbonyl group, an N-sulfosuccinimidyloxycarbonyl group, a halocarbonyl group, an isothiocyanate group, a vinyl group, a formyl group, a hydroxyl group, a mercapto group, a hydrazino group, an acylhydrazino group, an aminooxy group, a carbodiimide group or an acid anhydride residue; -A represents a chemical bond or a spacer group; -R5 and R6 are the same or different and represent a hydrogen atom; a halogen atom; an -OR4 group, wherein R4 represents a hydrogen atom, an aryl group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms; an aryl group; or an aliphatic hydrocarbon group having 1 to 6 carbon atoms optionally substituted by one or more groups selected from halogen atoms, aryl groups and hydroxyl groups; and -R10 represents a hydrogen atom, an aromatic group or an aliphatic hydrocarbon group having 1 to 6 carbon atoms.

10. The hydrogel according to claims 1 to 8, prepared by a method comprising the steps of: a) providing at least one polysaccharide or a salt thereof; b) providing at least one cross-linking agent or a salt thereof; c) preparing a cross-linking reaction medium comprising one or more polysaccharides, one or more cross-linking agents and a solvent, wherein the total amount of the cross-linking agent ranges from 0.001 mol to 0.03 mol per 1 mol of polysaccharide repeating unit, the duration of the reaction medium preparation step preferably not exceeding 5 hours; and d) placing the reaction medium obtained at the end of step c) under a pressure P and a temperature T, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P, preferably for a duration ranging from 1 week to 17 weeks.

11. The hydrogel according to claim 1 to 7 or 9, prepared by a method comprising the steps of: a) providing at least one polysaccharide or a salt thereof; b) providing at least one functionalizing agent as described in claim 9 or a salt thereof; c) preparing a cross-linking reaction medium comprising one or more polysaccharides, one or more functionalizing agents and a solvent, wherein the total amount of the functionalizing agents ranges from 0.001 mol to 0.50 mol per 1 mol of polysaccharide repeating unit, the duration of the reaction medium preparation step preferably not exceeding 5 hours; and d) placing the reaction medium obtained at the end of step c) under a pressure P and a temperature T, wherein the pressure P is less than or equal to atmospheric pressure, and the temperature T is greater than the temperature of the eutectic point of the reaction medium measured at the pressure P and less than the temperature of the freezing point of the reaction medium measured at the pressure P, preferably for a duration ranging from 1 week to 17 weeks.

12. A hydrogel according to any one of the preceding claims, wherein The cross-linked polysaccharide is hyaluronic acid, hyaluronate or a mixture thereof.

13. The hydrogel according to any one of the preceding claims, in sterile form.

14. The hydrogel according to any one of the preceding claims, which is in injectable form.

15. The hydrogel according to any one of the preceding claims, further comprising an anesthetic agent.

16. Cosmetic use of a hydrogel according to any of the preceding claims for preventing and / or treating alterations in the viscoelastic or biomechanical properties of the skin; to fill wrinkles, fine lines and scars; to reduce nasolabial folds and sadness lines; to increase the volume of the cheekbones, chin or lips or to reduce the appearance of wrinkles and fine lines.

Images