WO2025125597A1

WO2025125597A1 - Polysaccharide-based hydrogel comprising silicon

Info

Publication number: WO2025125597A1
Application number: PCT/EP2024/086276
Authority: WO
Inventors: Jimmy FAIVRE; François BOURDON
Original assignee: Teoxane SA
Current assignee: Teoxane SA
Priority date: 2023-12-15
Filing date: 2024-12-13
Publication date: 2025-06-19
Anticipated expiration: 2026-06-15

Abstract

The present disclosure relates to a polysaccharide-based hydrogel comprising silicon at a concentration of at least 15 ppm and to a process for the preparation thereof.

Description

POLYSACCHARIDE-BASED HYDROGEL COMPRISING SILICON

FIELD OF THE INVENTION

The present invention relates to a polysaccharide-based hydrogel comprising silicon and to a process for the preparation thereof.

BACKGROUND OF THE INVENTION

Polysaccharide hydrogels are used in various fields such as aesthetic, cosmetic and therapeutic fields. They may find applications in ophthalmology, periodontology, rheumatology or cosmetic surgery. For instance, hyaluronic acid hydrogels can be used as soft tissue fillers to correct volume defects such as wrinkles, scars or to increase the volume of soft tissues.

Today, there is a growing need for polysaccharide-based hydrogels capable of delivering additional beneficial effects, for instance capable to improve skin quality. In this context, silicon could be an element of choice.

Silicon (Si) is an abundant element on earth and is one of the most abundant trace elements in the human body. Numerous studies show that silicon, even in trace amounts, plays an important biological role, particularly in connective tissue architecture. Silicon would be important for optimal collagen synthesis and activation of hydroxylating enzymes, improving skin strength and elasticity.

However, due to their low solubility, most forms of silicon remain difficult to formulate in waterbased solutions and resultantly silicon is poorly bioavailable. For instance, orthosilicic acid is an aqueous soluble form of silicon. However, as orthosilicic acid is chemically unstable at physiological pH, it is rapidly converted, by polycondensation, into insoluble inorganic forms such as silica and silicates. The concentration of orthosilicic acid able to be dissolved in a waterbased solution remains quite low (<40 ppm) at room temperature, in pH conditions ranging or example from 4 to 9, preferably from 5 to 8, and in the absence of nucleation inhibitors or other stabilizers

Efforts have been directed to identify ways of increasing the solubility and stability over time of silicon in aqueous medium (e.g. silicon chemical modifications, use of stabilizing agents). However, the so far proposed solutions are for many not suitable for formulating injectable products and still do not allow to prepare polysaccharide-based hydrogel having high concentrations of silicon. Therefore, there remains a need to overcome the disadvantages of the state of the art by providing a process for preparing a polysaccharide-based hydrogel comprising a high concentration of a stable and highly assimilable bioavailable silicon. Advantageously, the process will allow preparing polysaccharide-based hydrogels having rheological properties suitable for the intended uses.

SUMMARY OF THE INVENTION

The invention relates to a process for preparing a polysaccharide-based hydrogel comprising at least 15 ppm of silicon as measured by ICP-OES. The process comprises the step of adding a supersaturated silicon solution in the preparation medium of the polysaccharide-based hydrogel or using a supersaturated silicon solution as the preparation medium of the polysaccharide-based hydrogel, the supersaturated silicon solution having a concentration of dissolved silicon of at least 40 ppm as measured by ICP-OES.

The invention relates to a polysaccharide-based hydrogel comprising silicon at a concentration of at least 40 ppm obtainable by the process according to the present invention and to a composition comprising thereof.

The invention relates to the cosmetic use of the polysaccharide-based hydrogel and compositions comprising thereof to prevent and/or treat alteration of the viscoelastic or biomechanical properties of the skin; to fill volumetric defects in the skin, in particular to fill wrinkles, fine lines and scars ; to attenuate nasolabial folds and bitterness lines; to increase the volume of cheekbones, chin or lips; to restore facial volumes, notably cheeks, temples, oval of the face, and around the eyes; to reduce the appearance of fine lines and wrinkles and their uses for filling and/or replacing tissue, especially soft tissue.

Further aspects of the invention are as disclosed herein and in the claims.

DEFINITIONS

“Silicon” or “Si” as used herein refers to the chemical element with the symbol Si and atomic number 14. It may also be referred, though rarely, as “silicium”.

“Silica” or “silicon dioxide” refers to the molecule SiO₂ and is one of the most abundant minerals on Earth. Silica can be found in various forms such as quartz, sand, and glass. Silica is also a significant component of many rocks and minerals. “Silicic acid” as used herein designates the soluble form of silica, either in its monomeric (orthosilicic acid) or dimeric form.

“Mesoporous silica” as used herein refers to silica with a specific pore size diameter. Pore diameter is defined as the average distance between pore walls. According to the IUPAC recommendations for characterization of porous solids, porous materials are classified as mesoporous when the pore diameter is between 2 and 50 nm (Loni 2014 Springer International Publishing Switzerland). This classification does not contain any information of pore morphology e.g. geometry, orientation, interconnectivity of pores, etc.

“Supersaturation” or “supersaturated solution” as used herein refers to a state in which a solution contains a higher concentration of a solute (for example silicon or silicic acid) than it can typically hold in equilibrium at a given temperature and pressure.

Because the solute is present in a greater amount than it would normally dissolve in the solvent under specific conditions, a supersaturated solution is not in its lowest energy state. Supersaturated solutions are considered metastable as they can spontaneously return to the stable state by precipitating the excess solute. Any disturbance (such as agitation or temperature or pH change) or introduction of nucleation sites can trigger the precipitation process, causing the solute to come out of the solution until the equilibrium concentration is reached.

Managing the supersaturated state requires thus careful control and manipulation of several factors, including the nature of the solvent system, temperature, pH, and the presence of stabilizing or inhibiting agents, to maintain the solution in its metastable condition, preventing premature precipitation and maximizing the potential for desired applications.

“Scaling” in the context of a supersaturated silicon solution, refers to the process of solid silica particles precipitating out of the solution and depositing onto surfaces (such as container walls, equipment surfaces or any other available nucleation sites) or forming aggregates. Scaling occurs when the concentration of dissolved silica in the solution exceeds its saturation limit at a given temperature and pH, leading to the spontaneous formation of solid silica particles. The scaling process can be detrimental in various applications, particularly in industrial settings, where the deposition of solid silica particles on equipment surfaces can lead to fouling, reduced efficiency, and increased maintenance costs.

“Nucleation” as used herein is the initial stage of crystallization or precipitation in a supersaturated solution. It refers to the formation of tiny solid clusters (nuclei) that act as the starting point for the growth of larger solid particles. In the context of an unstable supersaturated silicon solution, nucleation occurs when the concentration of dissolved silica exceeds its saturation limit, but the solution is unable to maintain its supersaturation state.

Nucleation is a critical step in the scaling process. Once nucleation occurs, solid silica particles start to form and grow, consuming the excess dissolved silica in the solution. This results in a reduction in the concentration of dissolved silicon, eventually leading to the solution returning to its equilibrium state or becoming undersaturated.

“PBS” or “phosphate buffered saline” is a buffer solution commonly used in biological and chemical applications due to its ability to maintain a stable pH. The pH of PBS is buffered from 6.8 to 7.8 at room temperature, for example buffered at 7.4 at room temperature.

The expression “% by weight”, “weight percent”, “w%”, “%w”, “%wt” or “wt%”, here and throughout the description unless otherwise defined, refers to the relative weight of the respective component based on the overall weight of the formulation.

The term "gel" refers to a polymer network that is expanded by a fluid. This means that a gel is made up of two media, one "solid" and one "liquid", dispersed in each other. The "solid" medium is made of long polymer molecules connected to each other by weak bonds (e.g. hydrogen bonds) or covalent bonds (cross-linking). The liquid medium consists of a solvent. A gel generally corresponds to a viscoelastic product which has a phase angle 5 of less than 90°, preferably less than or equal to 70°, more preferably less than or equal to 45°, at 1 Hz for a deformation of 0.1 % or a pressure of 1 Pa, preferably a phase angle 5 ranging from 2° to 45° or ranging from 20° to 45°.

The term "hydrogel" refers to a gel as defined above in which the liquid medium is predominantly water (e.g. at least 90%, in particular at least 95%, notably at least 97%, especially at least 98% by weight of the liquid medium is water) and having a pH ranging from

6.8 to 7.8.

The term "injectable hydrogel" refers to a hydrogel that can flow and be injected manually by means of a syringe fitted with a needle with a diameter ranging from 0.1 to 0.5 mm, for example a 32 G, 30 G, 27 G, 26 G, 25 G hypodermic needle. Preferably, an "injectable hydrogel" is a hydrogel exhibiting an average extrusion force of less than or equal to 25 N, preferably ranging from 5 to 25 N, still more preferably ranging from 8 to 15 N, when measured with a dynamometer, at a fixed speed of approximately 12.5 mm/min, in syringes of external diameter greater than or equal to 6.3 mm, with a needle of external diameter less than or equal to 0.4 mm (27 G) and length 1 ", at room temperature.

A "superficial application" refers to the administration of a composition, for example by mesotherapy, superficially into the skin, or onto the skin, for the treatment of superficial layers of the skin, the epidermis and the most superficial parts of the dermis, to reduce superficial wrinkles and/or improve the quality of the skin (such as its density or structure) and/or rejuvenate the skin.

A "median application" refers to the administration of a composition to the median part of the skin to treat the median layers of the skin, as well as to reduce median wrinkles.

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 contours. So-called "volumizing" hydrogels can typically be administered for deep application.

The term "polysaccharide" refers to a polymer composed of monosaccharides (preferably D- enantiomers) joined together by glycosidic bonds.

The term "repeating unit" of a polysaccharide refers to a structural unit made up of one or more (usually 1 or 2) monosaccharides, the repetition of which produces the complete polysaccharide chain. The "crosslinking rate" or “molar cross-linking rate”, expressed in %, refers to the ratio of the molar amount of crosslinking agent to the molar amount of polysaccharide repeating units introduced into the crosslinking reaction medium, expressed per 100 moles of polysaccharide repeating units in the crosslinking medium. For example, a crosslinking rate of 1 % means that there is one mole of crosslinking agent introduced into the reaction medium per 100 moles of polysaccharide repeating units.

A "cross-linked polysaccharide" refers to a polysaccharide modified during a cross-linking reaction.

A "non-crosslinked polysaccharide" refers to a polysaccharide that has not been modified with a crosslinking agent and has therefore not undergone a crosslinking reaction.

The term "cross-linking agent" refers to any compound capable of introducing cross-linking between different polysaccharide chains.

DESCRIPTION OF THE INVENTION

The inventors have developed a new process that allows preparing a polysaccharide-based hydrogel comprising at least 15 ppm silicon. More specifically, the inventors have developed a new process that allows preparing a polysaccharide-based hydrogel comprising a high concentration of silicon, i.e. a polysaccharide-based hydrogel comprising at least 40 ppm of silicon. The concentration of silicon in the polysaccharide-based hydrogel can be as high as 200 ppm.

Silicon concentrations disclosed herein are measured by ICP-OES. Concentrations measured by ICP-OES refer to concentrations of a soluble product.

The polysaccharide-based hydrogels obtained by the developed process contain high concentration of silicon that remains stable in physiological conditions (from 200 to 400 mOsm/kg; pH from 6.8 to 7.8).

Hence, the present invention relates to a process for preparing a polysaccharide-based hydrogel comprising at least 15 ppm of silicon, preferably at least 40 ppm of silicon. The process comprises the step of adding a supersaturated silicon solution in the preparation medium of the polysaccharide-based hydrogel or using a supersaturated silicon solution as the preparation medium of the polysaccharide-based hydrogel, the supersaturated silicon solution having a concentration of dissolved silicon of at least 40 ppm as measured by ICP-OES.

The present invention also relates to a polysaccharide-based hydrogel comprising at least 40 ppm of silicon obtainable by the process herein disclosed.

As indicated above, a supersaturated silicon solution is added during the preparation of the polysaccharide-based hydrogel: it is added in the preparation medium of the polysaccharide- based hydrogel. The addition of the supersaturated silicon solution which is as described herein below allows preparing polysaccharide-based hydrogel with advantageously high concentration of silicon (as measured by ICP-OES).

Alternatively, the supersaturated silicon solution may be used as the preparation medium of the polysaccharide-based hydrogel, i.e. it may be used to hydrate the polysaccharide when the polysaccharide is provided in dry form and is non-crosslinked.

Supersaturated silicon (Si) solution

The supersaturated silicon solution useful herein is a specialized solution in which the concentration of dissolved silicon exceeds its equilibrium solubility at a given temperature and under specific conditions. In such a solution, the dissolved silicon content surpasses the saturation point, making it thermodynamically unstable and prone to spontaneous precipitation or crystallization. One of the unique aspects of the supersaturated silicon solution useful herein is that the solution is stable even in the absence of stabilizers or crystallization inhibitors.

The supersaturated silicon solution comprises silicic acid in water-based solution.

The concentration of dissolved Si in the supersaturated silicon solution is at least 40 ppm, preferably at least 50 ppm, more preferably at least 60 ppm, or at least 70 ppm, or at least 80 ppm, or at least 90 ppm, or at least 100 ppm, or at least 110 ppm, or at least 120 ppm, or at least 130 ppm, or at least 140 ppm, or at least 150 ppm, or at least 160 ppm, or at least 170 ppm, or at least 180 ppm, or at least 190 ppm, or at least 200 ppm, as measured by ICP- OES.

In some preferred embodiments, the supersaturated silicon solution comprises from 40 to 200 ppm, or from more than 40 to 200, or from 40 to 170 or from 80 to 160 ppm, or from 100 to 160 ppm, or from 120 to 160 ppm, of silicon as measured by ICP-OES. The supersaturated silicon solution may further comprise a salt, more particularly a salt comprising a divalent cation, even more particularly a calcium salt or magnesium salt. The inclusion of calcium or magnesium salts drastically increases the production speed of supersaturated silicon solutions but has relatively little impact on their stability. Calcium and magnesium salts are thus considered processing aids, which improve the speed at which the supersaturated solutions are produced. They do not impact the stability and thus are not considered surfactants, emulgators or stabilizers.

The calcium salt can be selected from the list consisting of calcium acetate, calcium citrate, calcium lactate, calcium carbonate, calcium chloride, calcium gluconate, calcium hydroxide, calcium pantothenate, calcium phosphate, calcium stearate, calcium propionate, calcium butyrate, calcium formate, calcium sorbate and calcium benzoate. The calcium salt is preferably calcium acetate.

The magnesium salt can be selected from the list consisting of magnesium chloride, magnesium sulfate, magnesium nitrate, magnesium acetate, magnesium citrate and magnesium phosphate.

Other non-limiting salts that can be part of the supersaturated silicon solution are sodium chloride, potassium chloride, sodium sulfate, potassium sulfate, sodium nitrate, ammonium sulfate and lithium chloride.

The supersaturated Si solution useful herein is generally prepared in physiologically acceptable buffer solution. Examples of buffers include, but are not limited to, N-carbamoylmethyl taurine (CAS No: 7365-82-4), 3-[N,N-bis(hydroxyethyl)amino]-2- acid sodium salt hydroxypropane sulfonic acid (CAS No: 102783-62-0), 3-morpholino-2-hydroxypropane sulfonic acid (CAS No: 68399-77-9), 1 ,4-piperazinediethane sulfonic acid (CAS No: 5625- 37-6), 1 ,4-piperazine-N,N'- bispropane sulfonic acid) (CAS No: 5625-56-9), 2-hydroxy-3-

[tris(hydroxymethyl)methylamino]- acid 1 -propane sulfonic acid (CAS No: 68399-81-5), 2-[(2- hydroxy-1 ,1-bis(hydroxymethyl)ethyl)amino]ethane sulfonic acid (CAS No: 7365-44-8) , N- tris(hydroxymethyl)methylglycine (CAS No: 5704-04-1), 3-(N-morpholino)propane sulfonic acid (CAS No: 1132-61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1), bis(2- hydroxyethyl)amino-tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2- hydroxyethyl)taurine (CAS No : 10191-18-1), 4-(2-Hydroxyethyl)piperazine-1 -ethanesulfonic acid (CAS No: 7365-45-9), 1 ,4-Piperazinediethane sulfonic acid (CAS No: 5625-37- 6), 4-(2- hydroxyethyl)piperazine-1-(2-hydroxypropane-3-sulfonic acid) (CAS No: 68399-78-0), phosphate buffers such as PBS with a pH around pH physiological (CAS No: 7647-14-5, 7447- 40-7).

Preferably, the buffer is chosen from 3-(N-morpholino)propane sulfonic acid (CAS No: 1132- 61-2), tris(hydroxymethyl)aminomethane (CAS No: 77-86-1), bis (2-hydroxyethyl)amino- tris(hydroxymethyl)methane (CAS No: 6976-37-0), N,N-bis(2-hydroxyethyl)taurine (CAS No: 10191-18-1), acid 4 -(2-Hydroxyethyl)piperazine-1-ethane sulfonic acid (CAS No: 7365-45-9) and phosphate buffers such as PBS with a pH ranging preferably from 6.8 to 7.8 (CAS No: 7647-14-5, 7447-40 -7).

The supersaturated Si solution useful herein is preferably a water-based solution. A “waterbased solution” refers to a solution or homogeneous mixture wherein water serves as the solvent. In a water-based solution, organic solvents such as acetone or benzene are not present. The water-based medium may be water. In some embodiments, the supersaturated Si solution is buffered. The supersaturated Si solution may be preferably a phosphate buffered saline (PBS) solution, Tris or Bis-Tris buffer solution.

In some embodiments, the supersaturated silicon solution comprises dissolved silicon, at least one calcium salt and a water-based medium.

Dissolved silicon is known to be most stable at low pHs (e.g. lower than 4). Advantageously, the Si solutions useful in the present invention have elevated dissolved silicon with no stabilizers at higher pHs, e.g. ranging from 4 to 9. As a further advantage, the neutral and elevated pHs may be buffered.

The supersaturated silicon solution useful herein has typically a pH ranging from 4 to 9, or from 5 to 8, or from 5 to 7.8, or from 5 to 7.6, or from 5 to 7.5. At these pH the supersaturated silicon solution is stable, which means that no crystallization or scaling of the silicon occurs.

The concentration of particles present in the supersaturated silicon solution with a size of 0.50 pm or higher is typically at most 1 ppm, at most 0.8 ppm, at most 0.5 ppm, at most 0.4 ppm, at most 0.3 ppm, at most 0.2 ppm or at most 0.1 ppm relative to the supersaturated silicon solution. In some embodiments, the particles have a size of 0.45 pm or higher, 0.40 pm or higher, 0.35 pm or higher, 0.30 pm or higher, 0.25 pm or higher, 0.20 pm or higher or 0.1 pm or higher. In some embodiments, the concentration of particles present in the solution with a size of 0.20 pm or higher is at most 1 ppm, at most 0.8 ppm, at most 0.5 ppm, at most 0.4 ppm, at most 0.3 ppm, at most 0.2 ppm or at most 0.1 ppm relative to the supersaturated silicon solution.

The supersaturated silicon solution is typically stable (which means that no crystallization or scaling occurs) at a temperature ranging from 0 to 70°C, preferably from 4 to 50°C, more particularly from 4° to 30°C.

Known supersaturated silicon solutions can be obtained by adding stabilizers or chemical compounds that increase the solubility of silicon in water and stabilize silicon in the dissolved status. One of the advantages of the supersaturated Si solution useful herein is that it is provided in the absence of said compounds. The supersaturated Si solution comprises dissolved silicon without the need for stabilizers.

Thus, the supersaturated Si solution useful herein is preferably free of stabilizers, preferably free of stabilizers for silicon compounds (silicon stabilizers).

Phenols and quaternary ammonium salts are often used to stabilize silicon compounds, particularly monomeric and dimeric silicic acid as well as organosilicon compounds.

In some embodiments, the supersaturated Si solution useful herein is free of phenols and polyphenols. Phenol and other phenolic compounds can form soluble complexes with silicon species. Phenols, with their hydroxyl (-OH) groups, can coordinate with silicon, leading to the formation of stable phenol-silicon complexes.

In some embodiments, the supersaturated Si solution useful herein is free of quaternary ammonium salts, particularly choline salts. Amines, such as ammonia and organic amines, can form soluble complexes with silicon, increasing its solubility in various solvents.

Dissolved silicon can also be stabilized by adding chelating agents like EDTA (ethylenediaminetetraacetic acid), and oxalic acid. In some embodiments, the supersaturated silicon solution useful herein is free of chelating agents, more particularly free of EDTA (ethylenediaminetetraacetic acid) and/or and oxalic acid.

Some polymers or surfactants can stabilize silicon species in solution by forming micelles or providing a protective coating around the silicon particles, preventing their agglomeration or precipitation. In some embodiments, the supersaturated silicon solution is free of polymers or surfactants that stabilize silicon species in solution.

Because the supersaturated Si solution useful herein is typically free from compounds that stabilize Si in the dissolved status, adding one or more scaling nuclei to the supersaturated Si solution will initiate Si precipitation and thus a decrease of the dissolved Si content. The supersaturated Si solution useful herein may be characterized by a concentration of dissolved Si as measured by ICP-OES that decreases by at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 30% or at least 40%, or at least 50%, after a time period of 24 hours at 20°C, after introducing 100 mg of mesoporous silica particles / 100 ml of supersaturated silicon solution, wherein the mesoporous silica particles have a specific surface area of 750 m²/g, at pH 5. The introduction of mesoporous silica particles with a large and accessible surface area, leads to a relatively fast precipitation of supersaturated silicon monomers and dimers from the solution. Consequently, this destructive test acts as a means to measure the supersaturation of the silicon solution provided.

The size of the added particles is typically 0.45 pm or higher, 0.40 pm or higher, 0.35 pm or higher, 0.30 pm or higher, 0.25 pm or higher, 0.20 pm or higher or 0.1 pm or higher.

The supersaturated silicon solution useful herein is prepared by a method that allows obtaining a supersaturated silicon solution that is stable - hence, in which no scaling or crystallization or nucleation occurs - at room temperature, in pH conditions ranging from 4 to 9, in a water-based medium and in the absence of nucleation inhibitors or other stabilizers.

The method comprises the steps of: a. providing a mixture comprising mesoporous silica, preferably said mesoporous silica having a specific surface area of at least 500 m²/g; b. dissolving said mixture in a water-based medium in a range from 0.05 to 0.3% weight per volume (w/v), the water-based medium being at a temperature ranging preferably from 30 and 95°C; c. filtering the solution at a temperature ranging preferably from 30 to 95°C using a filter with a mesh size of at most 0.5 pm.

In preferred embodiments, the method comprises the steps of: a. providing a mixture comprising, or consisting of, from 20 to 95 w%, preferably from 20 to 40 w% mesoporous silica, preferably with a specific surface area of at least 500 m²/g; and from 80 to 5 w%, preferably from 80 to 60 w% of a salt comprising a divalent cation, preferably calcium or magnesium salt or mixture thereof; b. dissolving said mixture in a water-based medium in a range of from 0.05 to 0.3% weight per volume (w/v), the water-based medium being at a temperature ranging from 70 to 90°C; c. filtering the solution at a temperature ranging from 70 to 90°C using a filter with a mesh size of at most 0.5 pm.

The salts comprising a divalent cation increase the rate at which the mesoporous silica is broken down, in other words the dissolution rate. As a direct consequence, they reduce the process time or incubation time needed to obtain a certain silicon concentration; or increase the silicon concentration obtained at a set temperature and dissolution profile. Preferably, the mesoporous silica and salt comprising a divalent cation form 100 w%.

In some embodiments, the mixture of mesoporous silica and a divalent cation salt is composed of mesoporous silica and a divalent cation salt in a weight ratio of between 20:80, 25:75, 30:70, 35:65,40:60, 45:55, 50:50, 60:40, 70: 30, 75:25, 80:20, 85:15, 90:10, 95:5, preferably 20:80, 25:75, 30:70, 35:65, 40:60.

In some embodiments, the total Si content of the mixture ranges from 10 to 20 w%, or from 11 to 19 w%, or from 12 to 18 w%, or from 13 and 16 w% or around 14 w%.

The mesoporous silica useful to prepare the saturated Si solution has a specific surface area, calculated from nitrogen sorption isotherms at liquid nitrogen temperature (77K) by the Brunauer, Emmett and Teller (BET) theory, ranging from 10 to 1500 m²/g, preferably from 300 to 1250 m²/g, more preferably from 500 to 1000 m²/g, even more preferably from 500 to 800 m²/g. Higher surface area results in faster dissolution rates, and thus lower incubation times required to obtain a set silicon concentration and set temperature, or higher silicon concentration given a set dissolution time and temperature.

The mixture comprising the mesoporous silica, and the divalent cation salt when present, is dissolved in a water-based medium in a range of from 0.05 to 0.3% weight per volume (w/v). This means that from 0.05 gram to 0.3 gram of said mixture is dissolved in 100 ml of the waterbased medium. Before the mixture is added to the water-based medium, said medium is heated to a temperature preferably ranging from 30 to 95°C, preferably from 70 to 90°C, particularly from 72 to 85°C, more particularly from 75 to 80°C.

The dissolution temperature in step b) is preferably at least 30°C, preferably at least 40°C, preferably at least 50°C, more preferably at least 60°C or at least 65°C, or at least 70°C, or at least 75°C, or at least 80°C, or at least 85°C or at least 90°C. A higher temperature increases the equilibrium concentration. In other words, higher temperatures in dissolution step b), or in the incubation step between dissolution b) but prior to filtering c), result in a higher silicon concentration of the supersaturated Si solution. The maximum temperature should be below the boiling point of the solution, preferably at most 95°C.

The solution temperature may be gradually increased during the incubation. Advantageously, this allows a gradual increase of the dissolved Si concentration without the need to operate at the highest temperature throughout the full incubation period. Compared to operating at the maximum temperature obtained, this approach requires a longer incubation period but often lower overall energy and heating requirements.

The method may further comprise an incubation step before the filtering step c). The incubation step allows the mesoporous silica to break down to silicic acid and dissolve as completely as possible. The solution gradually reaches its equilibrium, having a longer incubation period allows for higher concentrations of silicon for any given temperature. The incubation step typically comprises incubating the obtained solution from step b) for at least 0.5 h at a temperature ranging from 30 to 95°C. Preferably the incubation step may comprise mixing or stirring. In some embodiments, the incubation step comprises incubating the obtained solution from step b) for at least 0.5 h, at least 1 h, at least 1 .5 h, at least 2 h, at least 3 h or at least 4 h at a temperature ranging from 70 to 90°C or from 72 and 85°C or from 75 to 80°C.

In step c), the solution should always be filtered at about the maximum temperature reached during dissolution step b) and the optional incubation step. Allowing the solution to cool prior to filtering all particles that can act as a nucleus from the solution results in precipitation, which is removed upon filtration, and thus lower final silicon concentrations in the resulting supersaturated Si solution. The filter used in step c) may have hydrophobic, hydrophilic or intermediate properties. In some embodiments, the filter is a hydrophobic filter, particularly a polytetrafluoroethylene (PTFE) membrane filter. In some embodiments, the filter is a hydrophilic filter, particularly a cellulose filter. In some embodiments, the filter is intermediate hydrophilic and hydrophobic properties, particularly a polyethersulfone (PES) or a polyvinylidene difluoride (PVDF) filter.

The filtering step is preferably performed at a temperature ranging from 72 to 85°C, more particularly from 75 to 80°C.

The filter used in the filtering step has typically a mesh size ranging from 0.1 to 0.5 pm, or from 0.2 to 0.45 pm, or from 0.3 to 0.4 pm, or from 0.15 to 0.3 pm or around 0.2 pm or around 0.45 pm.

The method typically comprises a cooling step after the filtering step c), wherein the cooling step comprises the cooling of the filtered solution to ambient temperature (temperature lower than or equal to 25°C, preferably from 15°C to 25°C). Preferably, no precipitation of silicon species to silica occurs during the cooling of the filtered solution. A second filtering step may then be performed after cooling the solution. The filtering of the solution may be performed with a mesh size of at most 0.5 pm, at a temperature lower than 25°C.

Hence, the method may further comprise the steps of: d) cooling the solution to a temperature lower than or equal to 25°C, and e) filtering the solution with a mesh size of at most 0.5 pm, at a temperature lower than 25°C.

The second filtering step e) may utilize a smaller mesh size compared to the first filtering step c). The second filtering step can benefit the long-term stability of the solution by removing residual particles.

Step d) may comprise at least one, preferably multiple cooling cycles to temperatures just above the freezing point. This improves the long-term stability of the supersaturated silicon solution. Without wishing to be bound by theory, by first reducing the temperature and promoting silicon precipitation, oligomeric silica with a particle size lower than the mesh size is allowed to grow and subsequently filtered from the solution effectively. This results in a method which removes particles just under the mesh size effectively, without the exponential increase in energy requirements by reducing the mesh size. This does come at the expense of a minor reduction in the final silicon concentration. The concentration of dissolved Si in the solution may then be determined, by ICP-OES.

Polysaccharides

The polysaccharide can be any polymer composed of monosaccharides joined together by glycosidic bonds, or mixtures thereof. Preferably, the polysaccharide is chosen from pectin and pectic substances; chitosan; chitin; cellulose and its derivatives; agarose; glycosaminoglycans such as hyaluronic acid, heparosan, dermatan sulfate, keratan sulfate, chondroitin and chondroitin sulfate; and mixtures thereof. Even more preferably, the polysaccharide is selected from hyaluronic acid, heparosan, chondroitin and mixtures thereof, even more preferably the polysaccharide is hyaluronic acid or a salt thereof, 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 particularly, hyaluronic acid is in its acid form or as a sodium salt (NaHA). The polysaccharide-based hydrogel can thus be a hydrogel based on hyaluronic acid and/or one of its salts.

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

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

A cross-linked polysaccharide can be prepared by any method known to the skilled person. For example, the cross-linked polysaccharide can be prepared as described in WO2010131175A1 , WO201277054A1 , WO2023/198917A1 , W02012077054A1 , W02010131175A1 ,

WO2023/198922A1 and WO2023/198920A1 .

The polysaccharide is preferably cross-linked by means of a cross-linking agent chosen from bi- or multi-functional epoxy or non-epoxy cross-linking agents. Examples of epoxy agents include 1 ,4-butanediol diglycidyl ether (BDDE), 1 ,2,7,8-diepoxy-octane, 1 ,2-bis(2,3- epoxypropyl)-2,3-ethane (EGDGE), polyethylene glycol)-diglycidyl ether (PEGDE), and mixtures thereof. 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, 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.

The cross-linked polysaccharide is preferably a cross-linked polysaccharide with a molar crosslinking rate of lower than or equal to 10%, preferably greater than 0 and lower than or equal to 6% or to 4% or to 2% or to 1 % (molar cross-linking rate = number of moles of cross-linking agent(s) per 100 moles of repeating unit of the polysaccharide(s)).

In particular, the cross-linked polysaccharide can be prepared by a process comprising the following steps:

(a1) preparing a cross-linking reaction medium comprising one or more polysaccharide(s), one or more cross-linking agent(s) and a solvent; and

(a2) reacting the reaction medium to obtain a cross-linked polysaccharide.

The previously described cross-linked and non-cross-linked polysaccharides are useful for preparing the hydrogels of the invention. The cross-linked or non-cross-linked polysaccharide, or the mixture thereof, will form the polymer network of the hydrogel. The hydrogel comprising a cross-linked or non-cross-linked polysaccharide, or a mixture thereof, can thus be said to be based on a cross-linked polysaccharide, or a non-cross-linked polysaccharide, or a mixture thereof. A hydrogel comprising, as the sole polysaccharide, a non-crosslinked polysaccharide, is prepared from a non-crosslinked polysaccharide. A hydrogel comprising a cross-linked polysaccharide as the sole polysaccharide is prepared from a cross-linked 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 hydrogel preparation.

Preparation of the polysaccharide-based hydrogel

The polysaccharide-based hydrogel may be prepared by different alternative methods.

The polysaccharide-based hydrogel can be prepared by adding the supersaturated silicon solution as described herein to the preparation medium of the polysaccharide-based hydrogel, said medium comprising a non-cross-linked polysaccharide or a cross-linked polysaccharide or a mixture thereof. The supersaturated silicon solution is possibly added to a hydrated non-cross-linked polysaccharide or to a hydrated cross-linked polysaccharide. In other words, it is typically added to a polysaccharide in the form of a gel.

The polysaccharide-based hydrogel can be prepared by using the supersaturated silicon solution as described herein as the preparation medium of the polysaccharide-based hydrogel. In particular, when the polysaccharide-based hydrogel comprises a non-cross-linked polysaccharide, the supersaturated silicon solution may be used as the hydration medium of the polysaccharide. In other words, the hyaluronic acid is hydrated in a medium comprising the supersaturated silicon solution or consisting of the supersaturated silicon solution.

In some alternative embodiments, when the polysaccharide-based hydrogel comprises a crosslinked polysaccharide, the supersaturated silicon solution may be added at the time the polysaccharide is cross-linked, i.e. in the cross-linking reaction medium. In other terms, the polysaccharide cross-linking may be performed in a reaction medium comprising the supersaturated silicon solution and then the polysaccharide-based hydrogel is prepared from the resulting cross-linked polysaccharide.

The preparation of a polysaccharide-based hydrogel from a cross-linked and/or non-cross- linked polysaccharide can be carried out in a conventional manner.

Thus, the preparation of a hydrogel comprising a cross-linked and/or non-cross-linked polysaccharide may comprise one or more of the following conventional steps:

- pH adjustment (1);

- Dilution (2);

- Purification (3);

- Addition of at least one additional component (4);

- Extrusion (5);

- Conditioning (6)

These steps are generally implemented after hydration of the hyaluronic acid (when provided in dry form).

These steps are well known to the skilled person and can be as described below. Some of these conventional steps can be carried out concomitantly. The conventional steps can be carried out in the following sequential order: pH adjustment (1) then dilution (2) then purification (3) then addition of an additional component (4) then extrusion (5). They may also be performed in a different order.

Conditioning (6) is typically performed after conventional steps (1) to (5).

The supersaturated silicon solution can be added/used before or after any of these conventional steps (1) to (5) or concomitantly.

Preferably, the addition of the supersaturated silicon solution is concomitant with the dilution step (2) or with the step of adding at least one additional component (4), in particular when the polysaccharide-based hydrogel comprises a cross-linked polysaccharide. In some embodiments, the addition of the supersaturated silicon solution is concomitant with the addition of an anesthetic agent. In some embodiments, the addition of the supersaturated silicon solution is concomitant with the addition of a lubricating agent. In some embodiments, the added supersaturated silicon solution may comprise other components, in particular a lubricating agent, for example non-crosslinked hyaluronic acid, non-crosslinked heparosan or a mixture thereof.

The skilled person will readily note that the amount of supersaturated silicon solution added in the preparation medium of the polysaccharide-based hydrogel will depend on many factors: the Si concentration of the supersaturated silicon solution; the desired Si concentration in the polysaccharide-based hydrogel; the step at which the supersaturated silicon solution is added in the preparation medium.

The skilled person will be able to adjust of the amount of supersaturated silicon solution added to the preparation medium with due consideration of these factors.

When the supersaturated silicon solution is added at the step (2) or (4), the concentration of dissolved silicon in the supersaturated solution silicon solution is preferably at least 2 times, or 3 times, or 4 times or 5 times, greater than the concentration of dissolved silicon in the polysaccharide-based hydrogel. In some embodiments, the supersaturated silicon solution allows to provide a high concentration of silicon in a small volume of solution thereby limiting the dilution of the gel during the preparation of the polysaccharide-based hydrogel. pH adjustment (1)

The polysaccharide-based hydrogel preparation process can include a step of adjusting the pH of the hydrogel to attain the desired pH (pH 6.8-7.8).

Dilution (2)

The polysaccharide-based hydrogel preparation process may include a step of dilution of the cross-linked and/or non-cross-linked polysaccharide. The dilution step makes it possible to adapt the polysaccharide concentration in the prepared hydrogel. In particular, an aqueous solvent is added to the cross-linked and/or non-cross-linked polysaccharide, for example, a physiological saline solution, possibly buffered by the presence of salts, such as phosphate salts. More particularly, the added aqueous solvent has a pH around physiological pH (6.8- 7.8). The polysaccharide concentration obtained following the dilution step advantageously varies from 1 mg/g to 50 mg/g hydrogel, more advantageously from 5 mg/g to 35 mg/g hydrogel, even more advantageously from 10 mg/g to 30 mg/g hydrogel.

Purification (3)

The polysaccharide-based hydrogel preparation process can include at least one purification step.

The purpose of the purification step is to remove any undesirable impurities. Such impurities may result from the cross-linking of the polysaccharide. Such impurities may include, for example, residual cross-linking agent, in particular of the epoxy type, which has not reacted. This step can also be used to perform a liquid exchange, such as a buffer exchange. The purification step can therefore be particularly useful when the hydrogel comprises a crosslinked polysaccharide.

Purification can be carried out by dialysis or filtration, for example by Dynamic Cross-flow Filtration (DCF).

Addition of additional components (4) The polysaccharide-based hydrogel preparation process can include one or more steps of adding at least one additional component. The additional component may be selected from anesthetic agents, antioxidants, lubricants, amino acids, peptides, proteins, vitamins (e.g. ascorbic acid and derivatives), minerals, nucleic acids, nucleotides, nucleosides, co-enzymes, adrenergic derivatives, sodium dihydrogen phosphate mono-hydrate and/or di-hydrate, sodium chloride and mixtures thereof.

Examples of lubricating agent include, but are not limited to, non-crosslinked polysaccharides, in particular non-crosslinked hyaluronic acid, non-crosslinked heparosan or mixture thereof. Examples of anesthetics include, but are not limited to, Ambucaine, Amoxecaine, Amylein, Aprindine, Aptocaine, Articaine, Benzocaine, Betoxycaine, Bupivacaine, Butacaine, Butamben, Butanilicaine, Chlorobutanol, Chloroprocaine, Cinchocaine, Clodacaine, Cocaine, Cryofluorane, Cyclomethycaine, Dexivacaine, Diamocaine, Diperodon, Dyclonine, Etidocaine, Euprocine, Febuerin, Fomocaine, GuafecaTnol, Heptacaine, Hexylcaine, Hydroxyprocaine, Hydroxytetracaine, Isobutamben, Leucinocaine, Levobupivacaine, Levoxadrol, Lidamidine, Lidocaine, Lotucaine, Menglytate, Mepivacaine, Meprylcaine, MyrtecaTne, Octacaine, Octodrine, OxetacaTne, OxybuprocaTne, ParethoxycaTne, ParidocaTne, PhenacaTne, Piperocaine, PiridocaTne, Polidocanol, Pramocaine, Prilocaine, Procaine, Propanocaine, Propipocaine, Propoxycaine, Proxymetacaine, Pyrrocaine, Quatacaine, Quinisocaine, Risocaine, Rodocaine, Ropivacaine, Tetracaine, Tolycaine, Trimecaine, and a salt thereof, in particular a hydrochloride salt, or a mixture thereof. Preferably, the hydrogel comprises an anesthetic agent as defined above, in particular lidocaine, mepivacaine or one of their salts such as the hydrochloride salt.

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

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

Examples of minerals include, but are not limited to, zinc salts (e.g. zinc acetate, particularly dehydrated or zinc citrate; preferably 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 pentahydrate), possibly in hydrated form, and mixtures thereof. Preferably, zinc citrate is chosen as an additional component.

Examples of nucleic acids include, but are not limited to, adenosine, cytidine, guanosine, thymine, thymidine, cytosine, derivatives thereof and mixtures thereof.

Examples of co-enzymes include, but are not limited to, coenzyme Q10, CoA, NAD, NADP, and mixtures thereof.

Examples of adrenaline derivatives include, but are not limited to, adrenaline, noradrenaline and mixtures thereof.

Extrusion (5)

The polysaccharide-based hydrogel preparation process can include one or more extrusion steps. This step makes it possible to obtain a more homogeneous hydrogel, in particular with an extrusion force that is as constant as possible, i.e. as regular as possible. For example, the extrusion step is performed by means of a sieve of which the perforations have a diameter of between 50 and 2000 pm. Those skilled in the art will know how to select the perforation diameter to suit the desired mechanical properties of the hydrogel.

Conditioning (6)

The polysaccharide-based hydrogel preparation process may additionally include a hydrogel conditioning step. Hydrogel conditioning is typically carried out in an injection device. Conditioning is preferably carried out just before the sterilization step. For example, the sterile polysaccharide-based hydrogel may take the form of an injection device pre-filled with the polysaccharide-based hydrogel, such as a syringe pre-filled with the polysaccharide-based hydrogel.

The process of the present invention typically includes a step of sterilizing the prepared polysaccharide-based hydrogel. Sterilization is preferably carried out by heat, for example in an autoclave. Sterilization is generally carried out by raising the temperature of the sterilization medium to a temperature known as the "plateau temperature", which is maintained for a set period of time known as the "plateau time". Sterilization is preferably carried out at a plateau temperature ranging from 121 °C to 135°C, preferably at a plateau time ranging from 1 minute to 20 minutes with F0 > 15. The sterilizing value F0 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 achieved by gamma, UV or ethylene oxide radiation. The polysaccharide-based hydrogel obtained by the process according to the invention typically has a pH ranging from 6.8 to 7.8 (physiological pH).

Polysaccharide based hydrogel

The polysaccharide-based hydrogel of the invention comprises silicon at a concentration of at least 40 ppm. It preferably comprises silicon at a concentration ranging from 40 to 200 ppm, preferably from more than 40 to 200 ppm or to 170 ppm or to 160 ppm or to 150 ppm. In some embodiments, the polysaccharide-based hydrogel of the invention, comprises from 40 to 120 ppm, more preferably from 40 to 70 ppm or from 40 to 60 ppm of Si. However, it shall be understood that polysaccharide-based hydrogels comprising silicon at concentrations lower than 40 ppm may be prepared by the process of the present invention.

The silicon in the polysaccharide-based hydrogel of the invention is in a soluble form. The silicon does not form any covalent bond with the polysaccharide in the hydrogel. The silicon remains in a free soluble form.

The polysaccharide-based hydrogel may be obtained by a process as described herein above. In particular, it may be obtained by a process comprising the step of adding a supersaturated silicon (Si) solution as disclosed herein in the preparation medium of the polysaccharide-based hydrogel or using a supersaturated silicon (Si) solution as disclosed herein as the preparation medium of the polysaccharide-based hydrogel.

The obtained hydrogel is a hydrogel based on a cross-linked polysaccharide or a non-cross- linked polysaccharide or a mixture thereof. The hydrogel obtained by the process of the present invention therefore comprises a cross-linked polysaccharide, or a non-cross-linked polysaccharide, or a mixture of a cross-linked polysaccharide and a non-cross-linked polysaccharide. It is understood that the cross-linked polysaccharide may be a mixture of crosslinked polysaccharides.

The polysaccharide-based hydrogel has a physiological pH, i.e. ranging from 6.8 to 7.8. The pH of the polysaccharide-based hydrogel is preferably greater than or equal to 6.9 and less than or equal to 7.5; 7.4; 7.3; 7.2; 7.1 or 7.

The polysaccharide-based hydrogel may comprise a cross-linked polysaccharide. A polysaccharide-based hydrogel comprising a cross-linked polysaccharide has advantageously a phase angle 5 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 5 ranging from 2° to 45° or ranging from 20° to 45°.

The polysaccharide-based hydrogel is preferably an injectable hydrogel, i.e. one which can flow and be injected manually by means of a syringe fitted with a needle of diameter ranging from 0.1 to 0.5 mm, for example a 32G, 30 G, 27 G, 26 G, 25 G hypodermic needle.

The polysaccharide-based hydrogel is preferably sterile.

The polysaccharide-based hydrogel 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 crosslinked and/or non-cross-linked polysaccharide, e.g. cross-linked and/or non-cross-linked hyaluronic acid), relative to the total weight of the hydrogel. Thus, when the hydrogel comprises, as the sole polysaccharide, a non-crosslinked polysaccharide, the polysaccharide-based hydrogel may comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of noncrosslinked polysaccharide (e.g. non-crosslinked hyaluronic acid), relative to the total weight of the hydrogel. When the polysaccharide-based hydrogel comprises, as sole polysaccharide, a cross-linked polysaccharide, the polysaccharide-based hydrogel may comprise from 0.1 to 5% by weight, preferably from 1 to 3% by weight, of cross-linked polysaccharide (e.g. cross-linked hyaluronic acid), relative to the total weight of the hydrogel. When the polysaccharide-based hydrogel comprises a mixture of cross-linked and non-cross-linked polysaccharide, the polysaccharide-based hydrogel may 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 (e.g. 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, based on the total weight of polysaccharide (e.g. hyaluronic acid) present in the hydrogel. The total polysaccharide concentration in the polysaccharide-based hydrogel advantageously ranges from 1 mg/g to 50 mg/g hydrogel, more advantageously from 5 mg/g to 35 mg/g hydrogel, even more advantageously from 10 mg/g to 30 mg/g hydrogel. Preferably the polysaccharide is hyaluronic acid, even more preferably sodium hyaluronate.

When the polysaccharide-based hydrogel comprises a cross-linked polysaccharide, the crosslinked polysaccharide preferably has a molar cross-linking rate of less than or equal to 10%. Preferably, the hydrogel comprises a crosslinked polysaccharide with a molar crosslinking rate greater than 0 and lower than or equal to 6%. Even more preferably, the polysaccharide-based hydrogel comprises a crosslinked polysaccharide with a molar crosslinking rate greater than 0 and lower than or equal to 4%. Even more preferably, the polysaccharide-based hydrogel comprises a crosslinked polysaccharide with a molar crosslinking rate greater than 0 and lower than or equal to 2%, preferably lower than or equal to 1 %, even more preferably lower 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 of the polysaccharide(s)).

In some embodiments, the polysaccharide-based hydrogel comprises an anesthetic agent. The anesthetic agent may be as described above, in particular the anesthetic agent may be mepivacaine, lidocaine or a salt thereof; more particularly in the form of a hydrochloride salt; preferably in amounts ranging 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.

Polysaccharide-based hydrogels of the invention are particularly useful for filling and/or replacing tissue, especially soft tissue. They can be injected in the tissue.

They can be injected using any of the methods known to the skilled person. In particular, they can be administered by means of an injection device suitable for intra-epidermal and/or intradermal and/or subcutaneous and/or supra-periosteal injection. In particular, the injection device may be chosen from a syringe, a set of micro-syringes, a wire, a laser or hydraulic device, an injection gun, a needle-free injection device, or a micro-needle roller. Polysaccharide-based hydrogels of the invention are preferably injected subcutaneously.

They may have deep, medium and/or surface applications.

They may have therapeutic and/or cosmetic and/or cosmeceutical applications.

In the cosmetics field, the polysaccharide-based hydrogels of the invention can be particularly useful for compensating for loss of tissue volume due to aging.

They can be used in the prevention and/or cosmetic treatment of an alteration in the surface appearance of the skin. For example, hydrogels can be used in the cosmetic field to prevent and/or treat alteration of the viscoelastic or biomechanical properties of the skin; to fill volumetric defects in the skin, in particular to fill wrinkles, fine lines and scars ; to attenuate nasolabial folds and bitterness lines; to increase the volume of cheekbones, chin or lips; to restore facial volumes, notably cheeks, temples, oval of the face, and around the eyes; to reduce the appearance of fine lines and wrinkles.

The process for preparing polysaccharide-based hydrogels of the invention is respectful of the properties of the hydrogels.

The present invention also relates to a composition comprising a polysaccharide-based hydrogel as described herein. The composition may be useful in therapeutic or cosmetic applications as disclosed herein above.

Embodiments of the present invention will now be described by way of the following examples which are provided for illustrative purposes only, and not intended to limit the scope of the disclosure.

EXAMPLES

A. Analytic methods

Measuring dissolved silicon

ICP-OES was used as an analytic method to obtain the concentration of dissolved silicon in a medium. ICP-OES is the appropriate measurement technique for measuring dissolved silicon content. It does not require chemical additives, has higher precision, accuracy and repeatability compared to silicomolybdic acid spectrophotometry, which cannot differentiate phosphor and silicon content.

ICP-OES is an analytical technique well-known by the skilled person in the art and used to determine the elemental composition of a wide range of samples. It is widely employed in various fields such as environmental analysis, metallurgy, pharmaceuticals, agriculture, and more. Briefly, a sample in liquid form is introduced into a high-temperature plasma in the ICP- OES instrument. The plasma is formed by ionizing an inert gas (usually argon) at extremely high temperatures (around 10,000 degrees Celsius). This results in the creation of a high- energy plasma gas consisting of positively charged ions and free electrons. The intense heat of the plasma causes the atoms and ions in the sample to become ionized (i.e. losing one or more electrons) and excited (i.e. electrons move to higher energy levels). As the excited ions and atoms in the plasma return to their ground state (lower energy levels), they emit light in the form of characteristic wavelengths or colors unique to each element. Each element emits light at specific wavelengths, forming a unique emission spectrum or “fingerprint” for that element. The emitted light is collected and passed through a spectrometer, which disperses the light into its constituent wavelengths. The spectrometer then measures the intensity of the emitted light at specific wavelengths. The intensity of the emitted light is directly proportional to the concentration of the corresponding element in the sample. By comparing the intensity of the emitted light at specific wavelengths with calibration standards of known elemental concentrations, the concentration of various elements in the sample can be determined accurately.

ICP-OES measures dissolved silicon, such as silicon in its monomeric and dimeric forms of dissolved silicic acid. It does not measure precipitated or gelled silicon, such as in the form of silica.

Characterization of all examples herein was done on an 5110 ICP-OES from Agilent to quantify Si content. Si calibration curve from 1 .56 to 25 ppm in ultra-pure water is performed with the appropriate dilution of the Si standard solution certified by supplier Chem-Lab. A clean plasma torch was used.

The samples at room temperature and lower are diluted with ultrapure water at a degree of dilution of 10:1 prior to analysis by ICP-OES.

The samples at temperatures above 30°C are filtrated on a 0.45 pm PTFE filter; subsequently diluted 10:1 with ultrapure water, and finally analyzed by ICP-OES at room temperature, unless otherwise specified. The filtration, prior to dilution, is performed to avoid changes in Si concentration due to precipitation or dissolution of precipitates due to dilution or temperature changes.

When the samples are hydrogels, 0.5 gram of hydrogel is diluted in 10g HNO₃ (70%) and 3.6 g HCI (30%) solution. The diluted hydrogel is aged for 10 min at 175 +/- 5°C in a micro-wave Anton Paar - Multiwave GO Plus (Ramp: 5.5 min, Temperature: 175 +/- 5°C, Hold: 4.5 min, Total duration: 10 min). After ageing, 10 ml of the solution are sampled and filtered on 0.45 pm PTFE filter. Samples are introduced in the autosampler of the ICP-OES for analysis.

The viscoelastic properties of the hydrogels were measured using a rheometer (DHR-2) having a stainless steel cone (1 ° - 40 mm) with cone-plane geometry and an anodized aluminum peltier plate (42 mm) (air gap 24 pm). 0.5 g of hydrogel is deposited between the peltier plate and said cone. Then a stress scan is performed at 1 Hz and 25°C. The elastic modulus G’, the viscous modulus G” and the phase angle 5 are reported for a stress of 5 Pa.

The stress at the intersection of G’ and G” T is determined at the intersection of the curves of the modules G’ and G” and is expressed in Pascal.

B. Materials

Phosphate-buffered saline (PBS): phosphate buffer

Hyaluronic acid 1 .5 MDa (fibers form)

BDDE

Lidocaine hydrochloride

NaOH 0.25N

HCI 1 N

IPM (calcium acetate/ mesoporous silica 700m²/g 70w%/30w%; 14 w% total Si content).

In all examples, ambient temperature means a temperature ranging from 21 to 25°C.

ss-linked

-based

160 silicon

Preparation of a phosphate buffered 160 ppm supersaturated silicon solution

500g of a phosphate buffer (PBS) were heated at a temperature of 80°C in a silicone oil bath and placed under magnetic stirring such as to form a vortex. 575 mg of IPM were then added to the phosphate buffer under magnetic stirring at 80°C. The mixture was maintained for 3 hours under stirring at 80°C and then hot filtered (80°C) using a 0.22 pm filtration unit. The obtained solution was then allowed to cool down to room temperature and stored.

The silicon content of the supersaturated silicon solution was 160 ppm as determined by ICP- OES. Preparation of the 20 mg/g non-cross-linked polysaccharide-based hydrogel

Hyaluronic acid (1.5 MDa) was added at room temperature to 30 g of the 160 ppm supersaturated silicon solution to prepare a 20mg/g non-cross-linked polysaccharide-based hydrogel. The resulting solution was placed under stirring at 150 rpm until complete dissolution of the hyaluronic acid.

After complete dissolution of hyaluronic acid, the obtained non-cross-linked polysaccharide- based hydrogel is packed into syringes and sterilized in an autoclave (plateau temperature ranging from 121 °C to 135°C with F0 > 15).

The resulting non-cross-linked polysaccharide-based hydrogel comprises 160 ppm of silicon as determined by ICP-OES, and is stable. No precipitation was observed after 2 months of storage at room temperature.

Example 2: preparation of a cross-linked polysaccharide-based hydrogel comprising 120 ppm silicon

Preparation of a phosphate buffered 160 ppm supersaturated silicon solution

The phosphate buffered 160 ppm supersaturated silicon solution was prepared as described previously (example 1).

Preparation of the cross-linked polysaccharide-based hydrogel

Hyaluronic acid (1.5 MDa) was dissolved in a 0.25 N sodium hydroxide solution in a pouch. The resulting 120 mg/g hyaluronic acid solution was kneaded with a paddle mixer until complete dissolution of hyaluronic acid.

BDDE (cross-linking rate: 8.25%) was then added to the 120mg/g hyaluronic acid solution and the mixture was then kneaded manually for 5 minutes and then kneaded with a paddle mixer for 15 minutes at 210 rpm. The mixture was then placed at 52°C for 3 hours.

The mixture was then diluted with the phosphate buffered 160 ppm supersaturated silicon solution to provide a cross-linked polysaccharide-based hydrogel having a hyaluronic acid concentration of 25 mg/g. The pH of the cross-linked polysaccharide-based hydrogel was measured and adjusted to about pH = 7 (addition of a solution 1 N HCI or 0.25N NaOH as appropriate). The cross-linked polysaccharide-based hydrogel was sieved at 710 m, packed into syringes and sterilized in an autoclave (plateau temperature ranging from 121°C to 135°C with F0 > 15). After sterilization, the cross-linked polysaccharide-based hydrogel had a physiological pH (pH = 6.8-7.8).

The resulting cross-linked polysaccharide-based hydrogel comprises 120 ppm silicon as determined by ICP-OES. It was found to be stable: no precipitation occurred upon and after sterilization and after a 6-month storage at room temperature. In addition, the cross-linked polysaccharide-based hydrogel was found to have rheological properties adapted to the intended uses.

¹ AG’ (%)= (G’ after sterilization - G’ before sterilization)/(G’ before sterilization) *100

² A 5 (%)= (5 after sterilization - 5 before sterilization)/( 5 before sterilization) *100

Example 3: preparation of a cross-linked and non-cross-linked polysaccharide-based hydrogel comprising 48 ppm silicon

Preparation of a phosphate buffered 160 ppm supersaturated silicon solution

Preparation of the polysaccharide-based hydroc/el

Hyaluronic acid (1.5 MDa) was dissolved in a 0.25 N sodium hydroxide solution comprising BDDE (cross-linking rate: 0.5%) in a pouch. The resulting 120 mg/g hyaluronic acid solution was kneaded with a paddle mixer for 1 hour at 210 rpm. The mixture was then placed at -20°C for 30 days. After thawing, the mixture was then diluted with phosphate buffer (PBS) to provide a polysaccharide-based hydrogel having a hyaluronic acid concentration of 23 mg/g, and a pH of about 7.

The mixture was then dialyzed for 48 hours at room temperature in PBS.

Then, a 160 ppm silicon supersaturated phosphate buffer solution comprising non-crosslinked hyaluronic acid (4 MDa) at a concentration of 23 mg/g was added to the cross-linked polysaccharide-based hydrogel (the non-crosslinked fraction represents 30 wt% relative to the total weight of the resulting cross-linked and non-cross-linked polysaccharide-based hydrogel).

A solution comprising lidocaine hydrochloride was also added to the cross-linked and non- cross-linked polysaccharide-based hydrogel (0.3 wt% relative to the total weight of the resulting cross-linked and non-cross-linked polysaccharide-based hydrogel).

After extrusion, the resulting cross-linked and non-cross-linked polysaccharide-based hydrogel was then sieved to micron size, packed into syringes and sterilized in an autoclave (plateau temperature ranging from 121 °C to 135°C with F0 > 15). After sterilization, the resulting polysaccharide-based hydrogel had a physiological pH.

The resulting polysaccharide-based hydrogel comprises 48 ppm silicon as determined by ICP- OES. It was found to be stable: no precipitation occurred upon and after sterilization and after a 6-month storage at room temperature. In addition, the polysaccharide-based hydrogel was found to have rheological properties adapted to the intended uses.

Claims

1. A process for preparing a polysaccharide-based hydrogel comprising at least 15 ppm of silicon as measured by ICP-OES, the process comprising the step of adding a supersaturated silicon solution in the preparation medium of the polysaccharide-based hydrogel or using a supersaturated silicon solution as the preparation medium of the polysaccharide-based hydrogel, the supersaturated silicon solution having a concentration of dissolved silicon of at least 40 ppm as measured by ICP-OES.

2. The process according to claim 1 , wherein the polysaccharide-based hydrogel comprises a cross-linked polysaccharide, a non-cross-linked polysaccharide or a mixture thereof.

3. The process according to claim 1 or 2, wherein the supersaturated silicon solution is prepared by a process comprising the step of: a) providing a mixture comprising mesoporous silica, preferably said mesoporous silica having a specific surface area of at least 500 m²/g; b) dissolving the mixture in a water-based medium in a range from 0.05 to 0.3% by weight per volume (w/v), the water-based medium being at a temperature ranging from 30 to 95°C; c) filtering the solution resulting from step b) at a temperature ranging from 30 to 95°C using a filter with a mesh size of at most 0.5 pm.

4. The process according to claim 3, wherein the process for preparing the supersaturated silicon solution further comprises an incubation step before the filtering step, the incubation step comprises incubating the obtained solution from step b) for at least 0.5 h at a temperature ranging from 30 to 95°C.

5. The process according to claim 3 or 4, wherein the process for preparing the supersaturated silicon solution further comprises a cooling step after the filtering step, wherein the cooling step comprises the cooling of the filtered solution to ambient temperature, wherein no precipitation of silica occurs.

6. The process according to any one of claim 3 to 5, wherein the mixture of step a) further comprises a salt comprising a divalent cation, preferably a calcium salt, more preferably selected from the group consisting of calcium acetate, calcium citrate, calcium chloride and calcium carbonate.

7. The process according to any one of claims 1 to 6, wherein the supersaturated silicon solution has a dissolved silicon concentration ranging from 40 to 200 ppm.

8. The process according to any one of claims 1 to 7, wherein the polysaccharide-based hydrogel comprises cross-linked polysaccharide and the supersaturated silicon solution is added in the preparation medium of the polysaccharide-based hydrogel after cross-linking of the polysaccharide.

9. The process according to any one of claims 1 to 7, wherein the polysaccharide-based hydrogel comprises non-cross-linked polysaccharide and the supersaturated silicon solution is used as the preparation medium of the polysaccharide-based hydrogel.

10. The process according to any one of claims 1 to 9, wherein the polysaccharide-based hydrogel comprises from 15 to 200 ppm, preferably from 40 to 200 ppm of silicon.

11 . The process according to any one of claims 1 to 10, wherein the supersaturated silicon solution is free from silicon stabilizers.

12. A polysaccharide-based hydrogel comprising silicon at a concentration of at least 40 ppm obtainable by the process according to any one of claims 1 to 11 .

13. A composition comprising a polysaccharide-based hydrogel comprising silicon at a concentration of at least 40 ppm obtainable by the process according to any one of claims 1 to 11.

14. Cosmetic use of a polysaccharide-based hydrogel according to claim 12 or of a composition according to claim 13 to prevent and/or treat alteration of the viscoelastic or biomechanical properties of the skin; to fill volumetric defects in the skin, in particular to fill wrinkles, fine lines and scars ; to attenuate nasolabial folds and bitterness lines; to increase the volume of cheekbones, chin or lips; to restore facial volumes, notably cheeks, temples, oval of the face, and around the eyes; to reduce the appearance of fine lines and wrinkles.

15. Polysaccharide-based hydrogel according to claim 12 or composition according to claim 13 for use for filling and/or replacing tissue, especially soft tissue.