WO2021115310A1

WO2021115310A1 - Cross-linked hyaluronic acid gel and preparation method therefor

Info

Publication number: WO2021115310A1
Application number: PCT/CN2020/134833
Authority: WO
Inventors: 黄兆辉; 张絮然; 解江冰
Original assignee: 爱博诺德(北京)医疗科技股份有限公司
Priority date: 2019-12-10
Filing date: 2020-12-09
Publication date: 2021-06-17

Abstract

The present invention relates to the field of medical biomaterials, in particular to a cross-linked hyaluronic acid gel as well as a method for preparing a cross-linked hyaluronic acid gel, the method at least comprising the following steps: (1) evenly mixing in a non-neutral environment an aqueous solution of hyaluronic acid and a cross-linking agent to form a mixed solution; (2) placing the mixed solution in a temperature that is lower than 0°C and greater than the eutectic point of the mixed solution so as to form a solid-liquid heterogeneous system; and (3) placing the solid-liquid heterogeneous system in a temperature that is lower than 0°C and greater than the eutectic point of the mixed solution so as to carry out a cross-linking reaction. Also disclosed is a composition comprising the foregoing cross-linked hyaluronic acid gel, and a use thereof.

Description

Cross-linked hyaluronic acid gel and preparation method thereof

Technical field

The present invention relates to the field of medical biological materials, in particular to cross-linked hyaluronic acid gel, and also relates to a method for preparing cross-linked hyaluronic acid gel. The present invention also relates to a composition comprising the cross-linked hyaluronic acid gel of the present invention, and its use.

Background technique

Hyaluronic acid is an important component of human skin, cartilage tissue and joint lubricating fluid. It is a chain polymer composed of (1-β-4)D-glucuronic acid and (1-β-3)N-acetamido The disaccharide units of glucose are repeatedly connected. Benefiting from its non-species difference, non-immunity and other advantages, natural hyaluronic acid is widely used in medical cosmetics, osteoarthritis treatment and eye surgery.

Natural hyaluronic acid has the shortcomings of short half-life in tissues, easy to be degraded and diffused, which greatly limits its application in soft tissue filling and other fields. Cross-linking and modifying natural hyaluronic acid with a chemical cross-linking agent to form a three-dimensional structure polymer gel containing chemical cross-linking bonds is a common method to solve the above-mentioned shortcomings of natural hyaluronic acid. However, when the cross-linked hyaluronic acid gel is administered to a living body, the residual cross-linking agent component after decomposition in the living body poses a certain safety hazard to the living body. Therefore, under the premise of ensuring the basic properties of cross-linked hyaluronic acid, it is of obvious significance to prepare cross-linked hyaluronic acid gel with as few chemical cross-linking agents as possible.

Cross-linked hyaluronic acid is widely used in medical injection to fill the cosmetic field, such as correcting wrinkles and wrinkles and increasing facial volume. Thanks to its non-immunogenicity and reversible filling process, cross-linked hyaluronic acid gel is considered by many to be one of the most ideal dermal fillers.

The elasticity, viscosity and cohesion of cross-linked hyaluronic acid are the key to affecting its plastic effect, maintenance time and injectability. The cross-linked hyaluronic acid gel with higher elasticity can provide stronger support for the upper covering tissue, and better resist wrinkles and skin sagging; viscosity and cohesion are the cornerstones to ensure the support of the gel. The high gel is not easy to shift and can be kept in the desired position after injection; the high cohesive gel can maintain the integrity of the gel without collapsing and spreading under the action of external force. Therefore, the lower the viscosity and cohesion, the shorter the maintenance time; but the injectability is inversely proportional to the elasticity, viscosity and cohesion of the product. The lower the elasticity, viscosity, and cohesion of the product, the lower the product The better the injectability.

In the application of cross-linked hyaluronic acid, there is a clear correlation between the performance requirements of the product and the injection filling site. The lower elasticity is suitable for filling in the superficial and middle dermis, and the higher elasticity is suitable for filling in the deep and subcutaneous dermis. If the parts used do not correspond, the plasticity of the product will be unnatural. However, the elasticity, viscosity and cohesion of cross-linked hyaluronic acid on the molecular scale are all derived from the chemical bonding and physical forces within and between the hyaluronic acid molecules. Under a certain formula and process parameters, the third There is a positive correlation between them. Therefore, when applied to shallow and middle fillings, if the elasticity of the gel is controlled, the viscosity and cohesive properties of the gel are always unsatisfactory. Gels tend to be easily displaced, collapsed or diffused, and the retention time is very short; if not controlled, the plastic effect is relatively rigid, and the gel is also difficult to push out from the fine needles.

Therefore, there is an urgent need for a cross-linked hyaluronic acid gel, which still has high viscosity and cohesion under low elastic modulus, and has good injectability, so as to achieve facial shaping and significantly improve the product The maintenance time.

At present, limited by the conventional process for preparing cross-linked hyaluronic acid, it is difficult to obtain the desired cross-linked hyaluronic acid gel that still has high viscosity and cohesion under low elastic modulus. The conventional process for preparing cross-linked hyaluronic acid is to mix hyaluronic acid and cross-linking agent in a non-neutral aqueous solution, and then form chemical bonds between the cross-linking agent and the groups on the hyaluronic acid polymer chain. Prepared together. The mainstream crosslinking agent on the market today is 1,4-butanediol diglycidyl ether (BDDE), and its marketed products include

Wait. A large number of related invention patents have been applied for in China, such as CN106589424, CN104086788, CN102660040, etc. However, from the technological point of view, these technological methods are roughly the same, and there is an obvious characteristic: one or more of the technological parameters used are more demanding. The reason is that this conventional chemical cross-linking process has a fundamental defect: in the reaction process, the non-neutral environment will cause the hyaluronic acid to continuously degrade. Therefore, in order to obtain cross-linked hyaluronic acid, the cross-linking reaction must proceed forward, that is, the rate of cross-linking network formation must be higher than the degradation rate of hyaluronic acid, which leads to stricter restriction on the reaction conditions, which is specifically expressed in the reaction required High concentration of hyaluronic acid, large amount of cross-linking agent, high reaction temperature and so on. The increase in the value of these process parameters will often degrade the properties of the gel. For example, increasing the concentration of hyaluronic acid will prolong the dissolution time, and increasing the reaction temperature will cause the degradation rate of hyaluronic acid to accelerate. These factors will all cause the degradation of hyaluronic acid. Speeding up will decrease the viscosity and cohesion of the gel; increasing the amount of crosslinking agent will increase the elasticity and decrease the viscosity and cohesion of the gel. This is in serious conflict with the desired higher viscosity and cohesion at low modulus of elasticity.

Therefore, in view of the fundamental defects of the conventional chemical cross-linking process methods, the existing process methods have the disadvantages of one or several harsh reaction conditions and the adverse effects caused by them, and they cannot effectively reduce the cross-linking process. The amount and residue of the coupling agent are currently not ideal for the optimization of conventional process methods. The industry also urgently needs a simple and convenient method for preparing cross-linked hyaluronic acid gel that can replace conventional processes, so as to obtain the required cross-linked hyaluronic acid gel (the cross-linked hyaluronic acid gel is in Under low elastic modulus, it still has high viscosity and cohesion, and has good injectability, which can achieve facial shaping and significantly increase the maintenance time of the product).

Summary of the invention

In order to solve the defects of the prior art, on the one hand, the present invention provides a novel preparation method of cross-linked hyaluronic acid gel. This method can not only significantly reduce the possibility that the chemical bonds within the molecular chain of hyaluronic acid will be severed, but also make the hyaluronic acid locally enriched during the reaction process to improve the crosslinking efficiency, so it can be used at a lower hyaluronic acid concentration , Cross-linking agent content and reaction temperature to obtain cross-linked hyaluronic acid products with good viscoelasticity, and reduce the possibility of cross-linking agent residue.

The present invention relates to a preparation method of cross-linked hyaluronic acid gel, which comprises the following steps:

(1) Mix the aqueous solution of hyaluronic acid, its metal salt, its derivative or its mixture with the cross-linking agent in a non-neutral environment to form a mixed solution;

(2) Put the mixed solution at a temperature lower than 0°C and higher than the eutectic point of the mixed solution to form a solid-liquid heterogeneous system;

(3) The solid-liquid heterogeneous system is placed at a temperature lower than 0° C. and higher than the eutectic point of the mixed solution for cross-linking reaction.

According to one embodiment of the present invention, the method of the present invention further includes the step of melting the solid phase in the solid-liquid heterogeneous system after the cross-linking reaction, and optionally subsequent steps of neutralization, purification and homogenization.

According to another embodiment of the present invention, the method of the present invention includes the following steps:

(1) Mix an aqueous solution containing a cross-linking agent and hyaluronic acid, its metal salt, its derivative or a mixture thereof uniformly in a non-neutral environment to form a mixed solution;

(2) Put the above-mentioned mixed solution at a temperature lower than 0°C and higher than the eutectic point of the mixed solution for a time sufficient to form a solid-liquid heterogeneous system to form a solid-liquid heterogeneous system;

(3) The heterogeneous system is placed at a temperature lower than 0° C. and higher than the eutectic point of the mixed solution for cross-linking reaction.

The method of the present invention also includes the step (4) of melting the solid phase in the heterogeneous system after the crosslinking reaction, and optionally further includes neutralization, purification and homogenization.

According to another embodiment of the present invention, the solid-liquid heterogeneous system is composed of ice crystals and a hyaluronic acid solution.

According to another embodiment of the present invention, in the cross-linked hyaluronic acid gel, in step (1), the cross-linking agent is selected from glycidyl ether, diepoxide, dicarbodiimide, divinyl sulfone, multifunctional Polyethylene glycol-based crosslinking agents and mixtures thereof. The crosslinking agent such as but not limited to 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polybutylene Glycol diglycidyl ether, 1,2-bis(2,3-epoxypropoxy)-2,3-ethylene, 1,2,7,8-diepoxyoctane, oxalic hydrazide, hexane Diamine, divinyl sulfone, and mixtures thereof.

According to another embodiment of the present invention, the said metal salt of hyaluronic acid refers to a salt formed by hyaluronic acid and metal ions, such as but not limited to sodium salt, potassium salt, magnesium salt, calcium salt of hyaluronic acid , Zinc salt and combinations thereof.

According to another embodiment of the present invention, the hyaluronic acid derivatives refer to polysaccharides containing hydroxyl groups, such as but not limited to those containing carboxymethyl cellulose, alginate, and chondroitin-4-sulfate. , Chondroitin-6-sulfate, xanthan gum, chitosan, pectin, agar, carrageenan or guar gum derivatives, and mixtures thereof.

According to another embodiment of the present invention, the concentration of the crosslinking agent is 0.05-20% w/w, preferably 0.5-10% w/w, more preferably 1-8% w/w, more preferably 2% to 6% w/ w.

According to another embodiment of the present invention, in the cross-linked hyaluronic acid gel, in step (1), the concentration of hyaluronic acid hyaluronic acid, its metal salts, its derivatives or mixtures thereof is at least 0.05% w/v And not higher than 50% w/v, preferably 0.1-30% w/v, even 0.5-10% w/v, especially 2-8% w/v, especially 4%-6% w/v.

According to another embodiment of the present invention, in the cross-linked hyaluronic acid gel, in step (1), the mass ratio of cross-linking agent to hyaluronic acid is 0.05-20% w/w, preferably 0.5-10% w/ w, more preferably 1 to 8% w/w, more preferably 2% to 6% w/w.

According to another embodiment of the present invention, the non-neutral environment can be an acidic environment, with a pH of 1 to 6, preferably 1 to 4, or an alkaline condition, with a pH of 9-14, preferably 11-14.

According to another embodiment of the present invention, in the cross-linked hyaluronic acid gel, the formation temperature of the solid-liquid heterogeneous system in step (2) is from the eutectic point of the mixed solution to 0°C, preferably from the eutectic point of the mixed solution to − 5°C, more preferably the eutectic point of the mixed solution is -10°C. The eutectic point refers to the temperature at which the water in the material (such as a mixed solution) completely freezes into ice crystals. The eutectic point of the mixed solution is determined by the physical properties and concentration of hyaluronic acid, its metal salts, its derivatives or their mixtures. For example, when the main component of the solution is hyaluronic acid and its concentration is 10%, the mixed solution’s The eutectic point range is about -30 to -80°C.

According to another embodiment of the present invention, the retention time of the crosslinked hyaluronic acid gel in step (2) is 1-24h, more preferably 2-12h, more preferably 4-8h.

According to another embodiment of the present invention, in the crosslinked hyaluronic acid gel, the crosslinking reaction temperature in step (3) is from the eutectic point of the mixed solution to 0°C, preferably from the eutectic point of the mixed solution to -5°C, More preferably, the eutectic point of the mixed solution is -10°C.

According to another embodiment of the present invention, in the cross-linked hyaluronic acid gel, the cross-linking reaction time in step (3) is within 100 days, preferably 1-28 days, more preferably 3-21 days, more preferably 5 ~14 days.

According to another embodiment of the present invention, the melting of the cross-linked hyaluronic acid gel in step (4) refers to converting ice crystals in a heterogeneous system into liquid water. For example, place the heterogeneous system at 25°C until the ice crystals melt naturally.

According to another embodiment of the present invention, in the cross-linked hyaluronic acid gel, the neutralization in step (4) refers to neutralizing the non-neutral environment in the cross-linking reaction to a neutral environment, for example, the pH is about 7. For example, the pH is 6.5 to 7.5, especially 6.8 to 7.2, especially 6.9 to 7.1.

According to another embodiment of the present invention, for the cross-linked hyaluronic acid gel, the purification in step (4) refers to reducing the residual amount of unreacted cross-linking agent in the gel to less than 2 μg/g. For example, but not limited to, the gel is dialyzed in a phosphate buffer with a neutral pH until the residual amount of cross-linking agent is less than 2 μg/g.

According to another embodiment of the present invention, in the cross-linked hyaluronic acid gel, the homogenization in step (4) refers to the cross-linked hyaluronic acid obtained in the cross-linking reaction by, for example, but not limited to, crushing and pressing The method of crushing and cutting is evenly dispersed into particles or solutions.

According to another embodiment of the present invention, when the concentration of the cross-linked hyaluronic acid gel is 10-30 mg/ml, its storage modulus G'value is between 10 Pa and 2000 Pa; G'value is the use of TA The DHR rotary rheometer is measured at a constant temperature of 25°C using a plate diameter of 40mm when the gap height is 1mm. Among them, each measurement is performed at a strain of 0.8% and a frequency of 1 Hz.

According to another embodiment of the present invention, when the concentration of the cross-linked hyaluronic acid gel is 10-30mg/ml, its loss modulus G" value is between 5Pa and 1000Pa; G" value is based on TA The DHR rotary rheometer is measured at a constant temperature of 25°C using a plate diameter of 40mm when the gap height is 1mm. Among them, each measurement is performed at a strain of 0.8% and a frequency of 1 Hz.

According to another embodiment of the present invention, when the concentration of the cross-linked hyaluronic acid gel is 10-30mg/ml, when the gel is extruded from a 27G needle, its extrusion force is at an extrusion rate of 30mm/min. The bottom is 5-50N.

According to another embodiment of the present invention, the cross-linked hyaluronic acid gel is a colorless and transparent gel.

According to another embodiment of the present invention, the median particle size of the cross-linked hyaluronic acid gel is between 100 μm and 1 mm.

According to another embodiment of the present invention, the pH of the cross-linked hyaluronic acid gel is in the range of 6.5-8.0.

According to another embodiment of the present invention, the protein content of the cross-linked hyaluronic acid gel is not more than 0.1% by mass.

According to another embodiment of the present invention, the heavy metal content of the cross-linked hyaluronic acid gel is not more than 5 μg/g.

According to another embodiment of the present invention, the residual amount of the cross-linking agent of the cross-linked hyaluronic acid gel is not more than 2 μg/g.

According to another embodiment of the present invention, the bacterial endotoxin content of the cross-linked hyaluronic acid gel is not more than 0.5 EU/mL.

The preparation method of the cross-linked hyaluronic acid gel of the present invention discards the conventional homogeneous cross-linking process, but adopts a solid-liquid heterogeneous system composed of ice crystals and hyaluronic acid solution. Joint process method.

Without being bound by any theory, it can be understood that during the cooling process of the method of the present invention, ice crystals appear in the aqueous solution of hyaluronic acid and gradually grow, so that the water in the liquid phase gradually decreases, and the concentration of hyaluronic acid gradually rises, and finally It constitutes a heterogeneous system of a solid phase formed by ice crystals and a liquid phase formed by hyaluronic acid and a small amount of water. In the heterogeneous system, on the one hand, the hyaluronic acid is locally enriched at a high concentration, and the concentration of hyaluronic acid has reached a level that cannot be achieved in a homogeneous environment, and the crosslinking efficiency is greatly improved. In addition, the formation of ice crystals greatly increases the concentration of hyaluronic acid in the liquid phase. The intra/inter-collision entanglement and hydrogen bonding of hyaluronic acid molecular chains are more frequent. The low temperature makes the hyaluronic acid molecular chains non-neutral. Under the conditions, the rate of breakage is reduced, and sufficient molecular chain length can maintain the hydrogen bond in/between molecular chains to the greatest extent, and at the same time, it provides sufficient conditions for the generation of intra/inter-chain entanglement of hyaluronic acid and thus significantly enhances it. On the other hand, the bonds between the molecular chains of hyaluronic acid are no longer easily cut off, which greatly inhibits the degradation of hyaluronic acid. The cross-linked network of the resulting gel is denser, and the viscoelasticity and resistance to enzymatic degradation are greatly improved; The linking agent can also react more fully, the utilization rate of the crosslinking agent is high, and the residual amount is low, which reduces the possibility of the crosslinking agent and the crosslinking agent whose one end is already in a bonded state and the other end is still a free state functional group. , Reduce the risk of use, break through the fundamental defects of conventional process methods.

The preparation method of the cross-linked hyaluronic acid of the present invention has low requirements for reaction conditions, a wider applicable process range, and more diversified properties of the cross-linked hyaluronic acid product obtained from the perspective of product performance. From the perspective of process conditions, the required concentration of hyaluronic acid is low, which reduces the difficulty of uniform mixing of hyaluronic acid raw materials; the amount of cross-linking agent required is low, which reduces the safety hazards of the product; the required reaction temperature is low, which avoids cross-linking. Co-agents such as thermal decomposition of BDDE and degradation of hyaluronic acid.

It can be seen that the preparation method of cross-linked hyaluronic acid of the present invention requires simple process conditions, is easy to realize and mass production, and meets the needs of industrial production scale.

The concentration of the active substance (such as a pH adjuster, such as sodium hydroxide) in the present invention refers to the mass ratio of the active substance to the volume of the solvent (such as water) (expressed in w/v%).

The concentration of hyaluronic acid, its metal salt, its derivative or its mixture in the present invention refers to the volume ratio of the mass of hyaluronic acid, its metal salt, its derivative or its mixture to the volume of the solvent (such as water) (w/v%).

The cross-linking agent concentration in the present invention refers to the quality of the cross-linking agent and the substance to be cross-linked (such as an aqueous solution of hyaluronic acid, which includes hyaluronic acid, hyaluronic acid metal salt, or hyaluronic acid derivatives or mixtures thereof ) Mass ratio (w/w%).

On the other hand, the present invention also relates to a cross-linked hyaluronic acid gel, especially an injectable cross-linked hyaluronic acid gel, which can be used for medical filling and beauty, for example, for filling and contouring the superficial and middle layers of the dermis. For shaping, especially in thinner skin areas, such as around the eyes, lips, etc., the gel contains hyaluronic acid-based gels, such as implants or fillers.

The gel of the present invention overcomes the shortcomings of known implants, still has high viscosity and cohesion under low elastic modulus, and has good injectability. When applied to the shallow and middle dermal fillings, the plasticity is natural and has a very natural plastic effect and better retention time, and can avoid or reduce the diffusion and displacement of the implant from the site where the implant is needed.

According to another specific embodiment, the cross-linked hyaluronic acid gel usually comprises a gel containing hyaluronic acid (HA) cross-linked with a cross-linking agent selected from the group consisting of: The agent is selected from glycidyl ether, diepoxide, dicarbodiimide, divinyl sulfone, multifunctional polyethylene glycol-based crosslinking agent and the like. The crosslinking agent such as but not limited to 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polybutylene Glycol diglycidyl ether, 1,2-bis(2,3-epoxypropoxy)-2,3-ethylene, 1,2,7,8-diepoxyoctane, oxalic hydrazide, hexane Diamine, divinyl sulfone or a combination thereof.

According to another specific embodiment, in the cross-linked hyaluronic acid gel, the molecular weight of the HA used for cross-linking is between 10 and 5 million, especially the molecular weight of the HA used for cross-linking is between 600 and 5 million. It can also be a composition with different molecular weights, preferably between 500,000 and 4 million, preferably between 600 and 5 million, especially between 700 and 4 million, or even between 800 and 3.5 million, especially It is between 1 million and 3.5 million, more particularly between 2 and 3 million, and more preferably between 500,000 and 2 million.

According to another specific embodiment, according to the crosslinked hyaluronic acid gel of the present invention, the mass ratio of crosslinker residues to hyaluronic acid residues in the gel is between 0.1-10%, preferably 0.5-5 %, more preferably 1-2.5%.

According to another specific embodiment, according to the cross-linked hyaluronic acid gel of the present invention, the elastic modulus of the gel is about 10 Pa to about 1000 Pa at 1 Hz; the viscous modulus of the gel is about 10 Pa to about 500 Pa; the tan δ value (the ratio of the viscous modulus to the elastic modulus) of the gel is about 0.4 to about 1.5 at 1 Hz.

According to another specific embodiment, the interface normal stress of the gel is between 1500 N/m ² and 7500 N/m ² .

According to another specific embodiment, the pushing force of the cross-linked hyaluronic acid gel is between 5 and 50N.

According to another specific embodiment, the cohesion of the cross-linked hyaluronic acid gel is about 2N to 10N.

According to another specific embodiment, the appropriate elastic modulus, viscous modulus, tanδ value, interface normal stress and push-out force are selected so that the cross-linked hyaluronic acid gel conforms to the following formula:

300m ^-2 <interface normal stress / push-out force <1500m ^-2

According to another specific embodiment, the elastic modulus of the gel is 10 Pa to 300 Pa at 1 Hz, preferably 50 Pa to 200 Pa. The modulus of elasticity is measured at a constant temperature of 25°C using a DHR rotational rheometer of TA, using a plate diameter of 40mm when the gap height is 1mm. Among them, each measurement is performed at a strain of 0.8% and a frequency of 1 Hz.

According to another specific embodiment, the viscous modulus of the gel is about 10 Pa to about 500 Pa at 1 Hz, preferably 80-100 Pa. The viscous modulus is measured using a DHR rotational rheometer of TA, using a plate diameter of 40mm when the gap height is 1mm, and measuring at a constant temperature of 25°C. Among them, each measurement is performed at a strain of 0.8% and a frequency of 1 Hz.

According to another specific embodiment, the tanδ value of the gel is from about 0.6 to about 1.5 at 1 Hz, and more preferably from about 0.6 to about 1 at 1 Hz. The tanδ value is measured at a constant temperature of 25°C using a DHR rotational rheometer of TA and a plate diameter of 40mm when the gap height is 1mm. Among them, each measurement is performed at a strain of 0.8% and a frequency of 1 Hz.

According to another specific embodiment, the cohesion of the gel is 2N to 10N, preferably 4N to 10N, more preferably about 4N to 8N.

Cohesion refers to the ability of a gel to maintain its shape. The cohesion of the gel of the present invention can be quantified as follows. Place 1 ml of gel sample on the flat surface of the rheometer to form a small conical pile. Load a movable upper plate with a diameter of 40 mm on the rheometer so that the sample is completely covered. Adjust the gap between the movable plate and the plane so that the movable plate just touches the gel but the normal stress is 0. During a period of time, move the plate slowly and steadily from this initial position until the gel just fills all the gaps between the movable plate and the plane and there is no overflow, record that the sample is applied in the normal direction at this time的力。 The force. This cohesion is used to determine the characteristic value of gel cohesion.

According to another particular embodiment, the interfacial gel method in stress between 3000N / m ² to 7500N / m ^2, more preferably greater than 4500N / m ² to 7500N / m ² between. The interface normal stress of the gel of the present invention is a characteristic value reflecting the cohesion of the gel, which can be quantified as follows: 1 ml of gel sample is placed on the plane of the rheometer to form a small conical pile. Load a movable upper plate with a diameter of r meters on the rheometer so that the sample is completely covered. Adjust the gap between the movable plate and the plane so that the movable plate just touches the gel but the normal stress is 0. During a period of time, move the plate slowly and steadily from this initial position until the gel just fills all the gaps between the movable plate and the plane and there is no overflow, record that the sample is applied in the normal direction at this time The force of is f Newton. Calculate the interface normal stress according to the following formula:

Normal interface stress = f/πr ² N/m ² .

According to another specific embodiment, the pushing force of the cross-linked hyaluronic acid gel is between 5N and 30N, more preferably between 5N and 15N. The pushing force of the gel of the present invention is a characteristic value reflecting the injectability of the gel, which can be quantified as follows: 1 ml of gel sample is put into a cylinder with a length of 80 mm and an inner diameter of 6.4 mm, and the front section of the cylinder is connected There is a 27G injection needle. When the gel is pushed out of the injection needle at a squeezing rate of 30 mm/min, the force required is recorded as the pushing force.

According to another specific embodiment, the HA concentration of the gel is greater than 16 mg/ml, preferably 20-25 mg/ml, more preferably 21-23 mg/ml.

According to another specific embodiment, the degree of crosslinking of the gel is between 2-10%, preferably 4-8%, more preferably 5-7%.

According to another specific embodiment, the cross-linked hyaluronic acid gel according to the present invention may also have at least one of the following:

-The protein content of the cross-linked hyaluronic acid gel is not more than 0.1% by mass;

-The heavy metal content of the cross-linked hyaluronic acid gel is not more than 5 μg/g;

-The residual amount of cross-linking agent of the cross-linked hyaluronic acid gel is not more than 2 μg/g; and/or

-The bacterial endotoxin content of the cross-linked hyaluronic acid gel is not more than 0.5 EU/mL.

The cross-linked hyaluronic acid gel of the present invention has the above-mentioned excellent properties. According to another embodiment of the present invention, the cross-linked hyaluronic acid gel of the present invention is specially prepared by the preparation method of the cross-linked hyaluronic acid gel of the present invention. . The preparation method of the novel cross-linked hyaluronic acid gel provided by the present invention can not only significantly reduce the possibility that the chemical bonds within the molecular chain of hyaluronic acid will be cut, but also make the obtained cross-linked hyaluronic acid gel more compact and have Greater cohesion, and the use of the least amount of cross-linking agent; at the same time, the hyaluronic acid can be locally enriched during the reaction process and the cross-linking efficiency can be improved. Therefore, it can be used at a lower hyaluronic acid concentration, cross-linking agent content and At the reaction temperature, a cross-linked hyaluronic acid product with better viscoelasticity is obtained, and the possibility of residual cross-linking agent is reduced.

The present invention also relates to the application of cross-linked hyaluronic acid gel in medical cosmetology, such as but not limited to the treatment of facial fine lines, facial sculpture, and correction of facial features.

According to another embodiment of the present invention, the cross-linked hyaluronic acid gel is used in surgery, soft tissue filling, wound hemostasis, wound healing, anti-scarring, scar repair, joint lubrication, and joint protection.

The present invention also relates to the cross-linked hyaluronic acid gel of the present invention, the composition containing the cross-linked hyaluronic acid gel of the present invention, or the medical device containing the cross-linked hyaluronic acid gel of the present invention in the treatment of medical beauty such as facial fine lines , Facial sculpture, facial feature correction, surgery or surgical treatment such as anti-adhesion and anti-scar application. Especially in soft tissue filling, wound hemostasis, wound healing, anti-scar, scar repair, joint lubrication, joint protection.

According to a specific embodiment of the present invention, the application of the cross-linked hyaluronic acid gel of the present invention in the dermis layer is more preferably the application in the superficial and middle layers of the dermis.

According to another specific embodiment of the present invention, the application of the cross-linked hyaluronic acid gel of the present invention on thinner skin areas, for example, includes around the eyes, lips and the like.

Specifically, the present invention relates to the following technical solutions:

1. A cross-linked hyaluronic acid gel, wherein the elastic modulus of the gel is about 10 Pa to about 1000 Pa at 1 Hz; the viscous modulus of the gel is about 10 Pa to about 500 Pa at 1 Hz; the gel The ratio of the viscous modulus to the elastic modulus tanδ value of the glue is about 0.4 to about 1.5 at 1 Hz; the cohesion is about 2N to 10N.

2. The cross-linked hyaluronic acid gel according to item 1, wherein the cross-linked hyaluronic acid gel conforms to the following formula:

300m ^-2 <interface normal stress / push-out force <1500m ^-2 .

3. The cross-linked hyaluronic acid gel according to item 1, wherein the tan δ value of the gel is about 0.6 to about 1.5, preferably about 0.6 to about 1, at 1 Hz.

4. The cross-linked hyaluronic acid gel according to item 1, wherein the cohesion of the gel is about 4N to about 10N, preferably about 4N to about 8N.

5. The cross-linked hyaluronic acid gel according to item 1, wherein the HA concentration of the gel is greater than 16 mg/ml, preferably 20-25 mg/ml, more preferably 21-23 mg/ml.

6. The cross-linked hyaluronic acid gel according to any one of the preceding items, wherein the elastic modulus is 10 Pa to 300 Pa, preferably 50 Pa to 200 Pa at 1 Hz.

7. The cross-linked hyaluronic acid gel according to any one of the preceding items, wherein the viscous modulus of the gel is about 10 Pa to about 500 Pa at 1 Hz, preferably 80-100 Pa.

8. The cross-linked hyaluronic acid gel according to any one of the preceding items, the degree of cross-linking is between 2-10%, preferably 4-8%, more preferably 5-7%.

9. The cross-linked hyaluronic acid gel according to any one of the preceding items, which has at least one of the following:

-The gel contains cross-linked hyaluronic acid (HA) cross-linked with 1,4-butanediol diglycidyl ether (BDDE);

10. The preparation method of cross-linked hyaluronic acid gel, especially the preparation method of cross-linked hyaluronic acid gel as described in any one of the preceding items, characterized in that it at least comprises the following steps:

(1) An aqueous solution of hyaluronic acid, or a metal salt of hyaluronic acid, or a hyaluronic acid derivative or a mixture thereof and a crosslinking agent are uniformly mixed in a non-neutral environment to form a mixed solution;

11. The method according to item 10, further comprising the step of melting the solid phase in the solid-liquid heterogeneous system after the crosslinking reaction, and optionally further comprising the steps of neutralization, purification and homogenization.

12. The method according to any one of the preceding items 10-11, which comprises the following steps:

(1) An aqueous solution containing a crosslinking agent and hyaluronic acid, or a metal salt of hyaluronic acid, or a hyaluronic acid derivative or a mixture thereof is uniformly mixed in a non-neutral environment to form a mixed solution;

(3) Place the heterogeneous system at a temperature lower than 0°C and higher than the eutectic point of the mixed solution for cross-linking reaction;

(4) Melt the solid phase in the heterogeneous system after the cross-linking reaction, and optionally neutralize, purify and homogenize.

13. The method according to any one of the preceding items 10-12, wherein the solid-liquid heterogeneous system is composed of ice crystals and a hyaluronic acid solution.

14. The method according to any one of the preceding items 10-13, wherein the crosslinking agent is selected from the group consisting of glycidyl ether, diepoxide, biscarbodiimide, divinyl sulfone, multifunctional polyethylene glycol-based crosslinking Coupling agents and mixtures thereof, such as 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polybutylene glycol Diglycidyl ether, 1,2-bis(2,3-epoxypropoxy)-2,3-ethylene, 1,2,7,8-diepoxyoctane, oxalic hydrazide, hexamethylene diamine , Divinyl sulfone, and mixtures thereof.

15. The method according to any one of the preceding items 10-14, wherein the metal hyaluronic acid salt is selected from the group consisting of sodium, potassium, magnesium, calcium, zinc, and combinations of hyaluronic acid.

16. The method according to any one of the preceding items 10-15, wherein the hyaluronic acid derivative is hyaluronic acid and selected from carboxymethyl cellulose, alginate, chondroitin-4-sulfate, chondroitin -Derivatives of 6-sulfate, xanthan gum, chitosan, pectin, agar, carrageenan, guar gum, and mixtures thereof.

17. The method according to any one of the preceding items 10-16, wherein the concentration of the crosslinking agent is 0.05-20% w/w, preferably 0.5-10% w/w, more preferably 1-8% w/w, and more It is preferably 2% to 6% w/w.

18. The method according to any one of the preceding items 10-17, wherein the concentration of the hyaluronic acid, its metal salt, its derivatives or mixtures thereof in step (1) is at least 0.05% w/v and not higher than 50% w/v, preferably 0.1 to 30% w/v, even 0.5 to 10% w/v, especially 2 to 8% w/v, especially 4% to 6% w/v.

19. The method according to any one of the preceding items 10-18, wherein the mass ratio of crosslinking agent to hyaluronic acid in step (1) is 0.05-20% w/w, preferably 0.1-15% w/w, more It is preferably 0.5 to 10% w/w, even 1 to 8% w/w, especially 2% to 6% w/w.

20. The method according to any one of the preceding items 10-19, wherein the non-neutral environment is an acidic environment, and the pH of the acidic environment is 1 to 6, preferably 1 to 4; or, the non-neutral environment It is an alkaline environment, and the pH of the alkaline environment is 9-14, preferably 11-14.

21. The method according to any one of the preceding items 10-20, wherein the formation temperature of the solid-liquid heterogeneous system in step (2) is from the eutectic point of the mixed solution to 0°C, preferably from the eutectic point of the mixed solution to − 5℃, even the eutectic point of the mixed solution to -10℃.

22. The method according to any one of the preceding items 10-21, wherein the holding time in step (2) is 1-24h, preferably 2-12h, especially 4-8h.

23. The method according to any one of the preceding items 10-22, wherein the crosslinking reaction temperature in step (3) is the eutectic point of the mixed solution to 0°C, preferably the eutectic point of the mixed solution to -5°C, or even mixing The eutectic point of the solution is -10°C.

24. The method according to any one of the preceding items 10-23, wherein the crosslinking reaction time in step (3) is within 100 days, preferably 1-28 days, more preferably 3-21 days, more preferably 5-14 days .

25. The method according to any one of the preceding items 10-24, wherein the melting refers to the conversion of ice crystals in a heterogeneous system into liquid water.

26. The method according to any one of the preceding items 10-25, wherein the neutralization refers to neutralizing the non-neutral environment in the cross-linking reaction to a neutral environment, that is, adjusting the pH to about 7, such as pH It is 6.5 to 7.5, especially 6.8 to 7.2, especially 6.9 to 7.1.

27. The method according to any one of the preceding items 10-26, wherein the purification refers to reducing the residual amount of unreacted cross-linking agent in the gel to within 2 μg/g.

28. The method according to any one of the preceding items 10-27, wherein the homogenization refers to uniformly dispersing the cross-linked hyaluronic acid obtained in the cross-linking reaction into particles or Solution.

29. The method according to any one of the preceding items 10-28, wherein the molecular weight of the HA used for cross-linking is 100,000 to 5 million, preferably 500,000 to 4 million, and more preferably 500,000 to 2 million.

30. A composition comprising the cross-linked hyaluronic acid gel according to any one of the foregoing items 1-9 or the cross-linked hyaluronic acid gel obtained according to any one of the foregoing items 10-29.

31. Medical equipment such as dressings, implants, and fillers, which comprise the cross-linked hyaluronic acid gel according to any one of the preceding items 1-9 or the obtained by the method according to any one of the preceding items 10-29 Cross-linked hyaluronic acid gel.

32. The cross-linked hyaluronic acid gel according to any one of the aforementioned items 1-9, the cross-linked hyaluronic acid gel obtained according to any one of the aforementioned items 10-29, the composition of item 30, or The medical device described in item 31 is used in medical beauty treatment such as facial fine lines treatment, facial sculpture, facial feature correction, surgery or surgical treatment such as anti-adhesion and anti-scarring.

33. The application according to item 32 above, which is used in soft tissue filling, wound hemostasis, wound healing, anti-scar, scar repair, joint lubrication, and joint protection.

34. The use according to any one of the preceding items 32-33, which is used in the dermis, particularly in the superficial and middle dermis.

35. The application according to any one of the preceding items 32-34, the application in the eye area and the lips.

Detailed ways

In order to better understand the present invention, further description will be given below in conjunction with specific embodiments.

The concentration of sodium hyaluronate in the present invention refers to the mass ratio of sodium hyaluronate to the volume of water (w/v%).

The concentration of sodium hydroxide in the present invention refers to the mass ratio of sodium hydroxide to the volume of water (w/v%).

The BDDE concentration in the present invention refers to the mass ratio of BDDE to sodium hyaluronate (w/w%).

Example 1:

The preparation method is as follows: sodium hyaluronate concentration 5w/v%, sodium hydroxide concentration 1w/v%, heterogeneous system formation temperature -20°C, holding time 2h, crosslinking reaction temperature -20°C, reaction time 3 days.

Prepare 20ml of 1w/v% sodium hydroxide aqueous solution, add the crosslinking agent BDDE according to the concentration shown in Table 1, and add 1g of sodium hyaluronate to it after mixing uniformly. The solution was stirred to homogeneity and kept at -20°C for 2 hours, and then placed in an environment of -20°C to react for 3 days. After the reaction is complete, place the product in room temperature environment until the ice crystals are completely melted, adjust the pH to neutral with hydrochloric acid solution, put it in PBS buffer for dialysis, purification, swelling, and mechanical homogenization to obtain a cross-linked sodium hyaluronate gel. It is 20mg/ml.

Comparative Example 1.1: In this comparative example, the gel was prepared under the same sodium hyaluronate concentration, cross-linking agent dosage and alkaline conditions as in Example 1, under the conventional process conditions of cross-linking reaction temperature of 40°C and cross-linking reaction time of 4 hours. glue. The preparation method is as follows, wherein the concentration of sodium hyaluronate is 5w/v%, the crosslinking reaction temperature is 40°C, and the crosslinking reaction time is 4h.

Prepare 20ml of 1w/v% sodium hydroxide aqueous solution, add the crosslinking agent BDDE according to the concentration shown in Table 1, and add 1g of sodium hyaluronate to it after mixing uniformly. After the solution was stirred to homogeneity, the mixture was placed in an oven at 40°C to react for 4 hours. After the completion of the reaction, the gel was adjusted to neutral pH with hydrochloric acid solution, put into PBS buffer for dialysis purification and mechanically homogenized to obtain a cross-linked sodium hyaluronate gel with a concentration of 20 mg/ml.

It must be pointed out that under such conventional reaction conditions, gels are formed only when the crosslinking agent concentration is 10w/w%, and the degradation rate of sodium hyaluronate at other lower crosslinking agent concentrations exceeds Because of the formation speed of the cross-linked network, no gel was formed after 4 hours of reaction. Continued extension of the reaction time will not change this result.

Without being bound by any theory, the reaction time of the control sample is 4h. This is because the reaction time of 4h is the closest to the highest degree of crosslinking under this reaction condition. If the reaction time continues to be prolonged, the reaction will be reduced due to the decrease of the crosslinking reaction rate. With hydrolysis as the main process, the performance of the product will decrease. If it is extended to 72h (the time used in the method of the present invention), the product may not be able to form a gel. On the contrary, the method of the present invention can prolong the reaction time (as described above to 72 hours), because the rate of the hydrolysis reaction in the method of the present invention is greatly suppressed, and the product can form a gel.

Comparative Example 1.2: In this comparative example, a gel was prepared under a higher sodium hyaluronate concentration and cross-linking agent dosage than in Example 1, under a cross-linking reaction temperature of 40° C. and a cross-linking reaction time of 4 hours. The preparation method is as follows, wherein the concentration of sodium hyaluronate is 10w/v%, the crosslinking reaction temperature is 40°C, and the crosslinking reaction time is 4h.

Prepare 20ml of 1w/v% sodium hydroxide aqueous solution, add the crosslinking agent BDDE according to the concentration shown in Table 1, and add 2g of sodium hyaluronate to it after mixing uniformly. After the solution was stirred to homogeneity, the mixture was placed in an oven at 40°C to react for 4 hours. After the completion of the reaction, the gel was adjusted to neutral pH with hydrochloric acid solution, put into PBS buffer for dialysis purification and mechanically homogenized to obtain a cross-linked sodium hyaluronate gel with a concentration of 20 mg/ml.

Comparative Example 1.3: An aqueous solution of sodium hyaluronate was prepared, in which the concentration of sodium hyaluronate was 20 mg/ml.

The cross-linked sodium hyaluronate gel prepared in Example 1, Comparative Example 1.1 and Comparative Example 1.2 was pushed out onto the stage of the rotary rheometer through a 27-gauge needle. The strain of the flat plate system was 0.8% and the frequency The viscoelasticity of the gel is characterized by 1 Hz oscillatory rheology, and the test results are listed in Table 1. The results show that under the process of the present invention (Example 1), the viscoelasticity of the gel has increased significantly compared with the sodium hyaluronate that has not been chemically crosslinked (Comparative Example 1.3). The increase in the increase in the increase rate also increases, which shows that under these process conditions, a denser cross-linked network is formed compared to the gel without chemical cross-linking. Under the process of the present invention, the cross-linking agent concentration can be selected in a wide range, and a cross-linked sodium hyaluronate gel with good viscoelasticity can be formed even at a very low cross-linking agent concentration.

Under the conventional process method (Comparative Example 1.1 and Comparative Example 1.2), the gel can be formed only when the concentration of the crosslinking agent reaches 10%, and the viscoelasticity, cohesion and interface normal stress of the obtained gel are all lower than the original The products obtained under the same or inferior conditions under the invented process method are precisely due to the fundamental defects of the conventional process method.

Table 1 Preparation process conditions of Example 1, Comparative Examples 1 to 3 and the properties of the gel obtained

*No gel formed, no viscoelastic characteristics and values.

Example 2:

The preparation method is as follows: cross-linking agent concentration 1.5w/w%, sodium hydroxide concentration 2w/v%, heterogeneous system formation temperature -25°C, holding time 1h, cross-linking reaction temperature -20°C, cross-linking reaction time 3 days .

Prepare 5 ml of 2w/v% sodium hydroxide aqueous solution, add the crosslinking agent BDDE according to the concentration shown in Table 2, and add sodium hyaluronate powder to the concentration shown in Table 2 after mixing. The solution was stirred to homogeneity and kept at -25°C for 1 h, and then placed at -20°C for 3 days. After the reaction is completed, the product is placed in a room temperature environment until the ice crystals are completely melted, the pH is adjusted to neutral with a hydrochloric acid solution, and then placed in PBS buffer for dialysis purification and mechanical homogenization to obtain a cross-linked sodium hyaluronate gel It is 20mg/ml. For the case where the concentration of sodium hyaluronate is 0.5w/v%, after the purification is completed, the gel is precipitated with alcohol, dried, and then re-dissolved in an appropriate amount of PBS to obtain a gel with a final concentration of 20 mg/ml.

Comparative Example 2: In this comparative example, the gel was prepared under the same sodium hyaluronate concentration, cross-linking agent dosage and alkaline conditions as in Example 2, under the conventional process conditions of cross-linking reaction temperature of 40°C and cross-linking reaction time of 4 hours. glue. The preparation method is as follows, wherein the crosslinking agent concentration is 1w/w%, the reaction temperature is 40°C, and the reaction time is 4h.

Prepare 5ml of 2w/v% sodium hydroxide aqueous solution, add crosslinking agent BDDE according to the concentration shown in Table 2, and add sodium hyaluronate according to the concentration shown in Table 2 after mixing. After the solution was stirred to homogeneity, the mixture was placed in an oven at 40°C to react for 4 hours. After the reaction is completed, the gel is adjusted to neutral pH with hydrochloric acid solution, put into PBS buffer solution for dialysis purification and mechanically homogenized. Under such conventional reaction conditions, the degradation rate of sodium hyaluronate surpassed its cross-linked network formation rate, so no cross-linked sodium hyaluronate gel was formed after 4 hours of reaction, and the reaction time will not change if the reaction time is extended. One result.

The cross-linked sodium hyaluronate gel prepared in Example 2 was pushed out onto the stage of the rotary rheometer through a 27-gauge needle, and the oscillating rheology of a flat system with a strain of 0.8% and a frequency of 1 Hz was used. The viscoelasticity of the gel was characterized, and the test results are listed in Table 2. The results show that under the process of the present invention (Example 2), the hyaluronic acid concentration can be selected in a wide range, and the cross-linking reaction can also occur at very low hyaluronic acid concentration to form a cross-linked sodium hyaluronate gel , The resulting gel has excellent properties.

However, under the conventional process method (Comparative Example 2), the cross-linked sodium hyaluronate gel cannot be formed under all reaction conditions, which is also caused by the fundamental defect of the conventional process method.

Table 2 Example 2, Comparative Example 2 Preparation process conditions and obtained gel viscoelasticity

*No gel formed, no viscoelastic characteristics and values.

Example 3:

The preparation method is as follows: sodium hyaluronate concentration 10w/v%, sodium hydroxide concentration 1w/v%, crosslinking agent BDDE concentration 1w/w%, heterogeneous system formation temperature -30℃, holding time 2h, crosslinking reaction time 3 days.

Prepare 10ml of 1w/v% sodium hydroxide aqueous solution, add 0.01g of cross-linking agent BDDE, mix well and add 1g of sodium hyaluronate. The solution was stirred to homogeneity and kept at -30°C for 2 hours, and then placed at the temperature in Table 3 to react for 3 days. After the reaction is completed, place the product in room temperature environment until the ice crystals are completely melted, adjust the pH to neutral with hydrochloric acid solution, put it in PBS buffer for dialysis purification and mechanical homogenization to obtain a cross-linked sodium hyaluronate gel with a concentration of 20mg/ml.

The cross-linked sodium hyaluronate gel prepared in Example 3 was pushed out onto the stage of the rotary rheometer through a 27-gauge needle, and the flat plate system was used for an oscillatory rheology with a strain of 0.8% and a frequency of 1 Hz. The viscoelasticity of the gel was characterized, and the test results are listed in Table 3. The results show that cross-linked sodium hyaluronate gel can be formed under these conditions, and the gel properties obtained are good.

Table 3 Example 3 Preparation process conditions and obtained gel viscoelasticity

Example 4:

The preparation method is as follows: sodium hyaluronate concentration 3w/v%, sodium hydroxide concentration 1w/v%, crosslinking agent BDDE concentration 2w/w%, heterogeneous system formation temperature -10℃, holding time 4h, crosslinking reaction temperature -20°C.

Prepare 40ml of 1w/v% sodium hydroxide aqueous solution, add 0.024g of cross-linking agent BDDE, mix well and add 1.2g of sodium hyaluronate. Stir the solution until it is uniform and place it at -10°C for 4 hours, and then place it at -20°C for the time listed in Table 4. After the reaction is completed, place the product in room temperature environment until the ice crystals are completely melted, adjust the pH to neutral with hydrochloric acid solution, put it in PBS buffer for dialysis purification and mechanical homogenization to obtain a cross-linked sodium hyaluronate gel with a concentration of 20mg/ml.

The cross-linked sodium hyaluronate gel prepared in Example 4 was pushed out onto the stage of the rotary rheometer through a 27-gauge needle, and the flat plate system was used for an oscillatory rheology with a strain of 0.8% and a frequency of 1 Hz. The viscoelasticity of the gel was characterized, and the test results are listed in Table 4. The results show that under the process of the present invention, the viscoelasticity of the obtained gel can gradually increase with the extension of the cross-linking time and remain stable after 14 days after the cross-linking agent is basically consumed. This shows that under the process of the present invention, the intra-chain bonds of sodium hyaluronate will not be severed, the chemical cross-linking reaction can also proceed smoothly, and the gel properties obtained are better.

Table 4 Example 4 Preparation process conditions and the resulting gel viscoelasticity

Example 5:

The preparation method is as follows: sodium hyaluronate concentration 4w/v%, crosslinking agent BDDE concentration 1w/w%, heterogeneous system formation temperature -15°C, holding time 3h, crosslinking reaction temperature -20°C.

Prepare 20 ml of 1% sodium hydroxide aqueous solution, add 0.008 g of cross-linking agent BDDE, mix well and add 0.8 g of sodium hyaluronate. The solution was stirred until it was uniform and kept at -15°C for 3h, and then placed at -20°C for the time listed in Table 3. After the reaction is completed, the product is placed in a room temperature environment until the ice crystals are completely melted, the pH is adjusted to neutral with a hydrochloric acid solution and mechanically homogenized to obtain a cross-linked sodium hyaluronate gel with a concentration of 20 mg/ml. In order to study the influence of different reaction times on the residual amount of the gel cross-linking agent of the invention, the gel obtained in this example was not purified.

The cross-linked sodium hyaluronate gel prepared in Example 5 refers to the industry standard YY/T0962-2014 "Cross-linked sodium hyaluronate gel for plastic surgery" in Appendix F.2 Gas Chromatography to determine the preparation prepared in Example 5. The test results of the residual amount of BDDE in the cross-linked sodium hyaluronate gel are listed in Table 5. The results show that with the extension of the reaction time, the residual amount of cross-linking agent in the gel gradually decreases, and it can be reduced to the industry standard YY/T0962-2014 "Cross-linked sodium hyaluronate gel for plastic surgery" after 14 days of reaction time 》The residual amount and method quantification limit (2μg/g) specified in the following. This shows that under the process method of the present invention, the chemical cross-linking reaction can be carried out more fully and the utilization efficiency of the cross-linking agent is high. After 14 days of reaction, the cross-linking agent has basically reacted completely, which is consistent with the data in Example 4.

Table 5 Example 5 Preparation process conditions and the resulting gel crosslinking agent residual amount

*The residual amount is lower than the residual amount and method quantification limit specified in YY/T0962-2014.

Example 6:

This example evaluates the enzymatic resistance of the cross-linked gel prepared under the process of the present invention. The concentration of the cross-linking agent BDDE is 1.5w/w%, the formation temperature of the heterogeneous system is -20°C, and the retention time is 2h. The cross-linking reaction temperature is -20°C, and the cross-linking reaction time is 5 days.

The preparation method is as follows:

Prepare 10ml of 1% sodium hydroxide aqueous solution, add the crosslinking agent BDDE according to the concentration shown in Table 6, and add sodium hyaluronate according to the concentration shown in Table 6 after mixing. After the solution was stirred to homogeneity, the mixture was placed at -20°C for 2 hours, and then transferred to -20°C for 5 days. After the reaction is completed, place the product in room temperature environment until the ice crystals are completely melted, adjust the pH to neutral with hydrochloric acid solution, put it in PBS buffer for dialysis purification and mechanical homogenization to obtain a cross-linked sodium hyaluronate gel with a concentration of 20mg/ml.

Comparative Example 6: In this comparative example, when the concentration of sodium hyaluronate is 10w/v%, the concentration of crosslinking agent is 10w/w%, the crosslinking reaction temperature is 40℃, and the crosslinking reaction time is 4h. The gel's resistance to enzymatic hydrolysis. The preparation method is as follows:

Prepare 10ml of 1w/v% sodium hydroxide aqueous solution, add 0.0125g of cross-linking agent BDDE, mix well and add 1g of sodium hyaluronate. After the solution was stirred to homogeneity, the mixture was placed in an oven at 40°C to react for 4 hours. After the completion of the reaction, the gel was adjusted to neutral pH with hydrochloric acid solution, put into PBS buffer for dialysis purification and mechanically homogenized to obtain a cross-linked sodium hyaluronate gel with a concentration of 20 mg/ml.

The enzymatic hydrolysis process is achieved by HAase-B, a hyaluronidase derived from Bacillus. Under suitable conditions, the enzyme can completely degrade HA into a single unsaturated disaccharide. The degradation product has strong UV absorption at a wavelength of 232nm. Therefore, the absorbance of the solution and the degradation end point can be measured after different enzymatic hydrolysis times. The absorbance of the solution reflects the degree of degradation of the gel.

Put 0.5g of the cross-linked sodium hyaluronate gel prepared in Example 6 and Comparative Example 6 into a sterile 24-well plate, and add 3ml of hyaluronidase solution (HAase-B, 200IU/ ml). Place the 24-well plate in a constant temperature bath at 42°C for 4.5 hours, then take 50μl of the supernatant and dilute it to 3ml, and measure the wavelength absorption at the UV 232nm wavelength. The absorbance no longer changes after the incubation for 24h, so the absorbance at 24h incubation is taken as the degradation end point, and the percentage of the absorbance at the degradation end point at 4.5h is taken as the enzyme degradation rate. The lower the enzymatic degradation rate, the better the resistance to enzymatic degradation of the gel, and the longer the filling effect of the gel in the body. The test results are listed in Table 6. The results show that under the process of the present invention, even if the concentration of the crosslinking agent used is much lower than that of the conventional process, the anti-enzymatic hydrolysis of the gel obtained after full reaction still shows a great advantage compared with the conventional process. Sex.

Table 6: Preparation process conditions of Example 6 and Comparative Example 6 and the anti-enzymatic hydrolysis of the gel obtained

Example 7:

The preparation method is as follows: prepare 20 ml of 1% sodium hydroxide aqueous solution, and add 1 g of sodium hyaluronate. Stir the solution until it is uniform and place it at -25°C for 2h to form a solid-liquid heterogeneous system. Subsequently, the solid-liquid heterogeneous system was placed in an environment of -20°C for a period of time, as shown in Table 7, and then the gel was adjusted to neutral pH with hydrochloric acid solution to obtain a sodium hyaluronate gel with a concentration of 20 mg/ ml.

Comparative Example 7: This comparative example investigates the influence of conventional processing methods on the molecular weight of sodium hyaluronate.

The preparation method is as follows:

Prepare 20 ml of 1% sodium hydroxide aqueous solution, and add 1 g of sodium hyaluronate. After the solution was stirred to uniformity, the mixture was placed in an environment of 40°C for the specific time as shown in Table 8. Then the gel was adjusted to neutral pH with hydrochloric acid solution to obtain a sodium hyaluronate gel with a concentration of 20 mg/ml .

The weight average molecular weight of the sodium hyaluronate prepared in Example 7 and Comparative Example 7 was measured by gel permeation chromatography. The results show that under the process of the present invention, even after maintaining for 3 days, the molecular weight of hyaluronic acid is still much larger than the molecular weight of hyaluronic acid in 1 day under conventional process conditions, while maintaining 2% under conventional process conditions. Days and 3 days later, the sodium hyaluronate molecule has been completely sheared into small molecules, which has been below the lower limit of detection, indicating that under the solid-liquid heterogeneous system, the intra-chain bonding of hyaluronic acid is not easy to be cut, and the alkali of hyaluronic acid Degradation is significantly inhibited.

Table 7 Example 7 and Comparative Example 7 The molecular weight distribution of sodium hyaluronate after different temperature and time treatments

In summary, the cross-linked network of the gel product of the present invention is denser, resulting in higher cohesion, excellent viscoelasticity, more diversified performance, and good enzymolysis resistance. The cross-linking agent reacts more fully, which can reduce the amount of cross-linking agent and reduce the residue of the cross-linking agent. It can achieve high viscosity and cohesion under low elastic modulus. The required process conditions are simple, easy to realize and mass production, and meet the needs of industrial production scale.

The following examples evaluate the properties of cross-linked hyaluronic acid gel products.

Example 8:

In this embodiment, the elastic modulus and viscous modulus of the cross-linked sodium hyaluronate gel at 1 Hz are 105 and 84 Pa, respectively, the tanδ value of the gel is 0.8, and the cohesion of the gel is 4.56N, the interface normal stress of the gel is 3631N/m ² , the pushing force of the gel is 8.3N, and the interface normal stress/pushing force of the gel is 437 m ^-2 .

Comparative Example 8:

The elastic modulus and viscous modulus of the cross-linked sodium hyaluronate gel at 1 Hz are 231 and 30 Pa, respectively, the tanδ value of the gel is 0.13, the cohesive force of the gel is 1.89N, and the coagulation The interface normal stress of the glue is 1505 N/m ² , the pushing force of the gel is 20 N, and the interface normal stress/ pushing force of the gel is 75 m ^-2 .

The following examples evaluate the filling of cross-linked hyaluronic acid gels.

Example 9: Injection and filling of the gel in the lacrimal trough and suborbital area

The elastic modulus and viscous modulus at 1 Hz made of hyaluronic acid of non-animal origin are 105 and 84 Pa, tanδ value is 0.8, cohesion force is 4.56N, interface normal stress is 3631N/m ² , pushing force The cross-linked hyaluronic acid gel (20mg/ml) with 8.3N and interface normal stress/extruding force of 437m ^-2 was put into a 1ml sterile glass syringe, and a 27G1/2 straight sharp needle was placed in the tear groove and The infraorbital dermis is injected from the superficial layer to the middle layer. Before the injection, the patient was given preoperative local anesthesia or nerve block anesthesia. Use retrograde injection or microsphere filling for injection, depending on the severity of the volume loss and the degree of zygomatic protrusion. If the bone surface is flat and the area of volume loss is extensive, needle withdrawal injection or microball injection can be used to avoid irregular contours. After completing the injection, gently massage the treatment area to keep it in line with the contours of the surrounding tissues. If necessary, apply ice temporarily to reduce redness and swelling.

By applying the cross-linked sodium hyaluronate gel of the present invention, it is observed that a satisfactory filling effect in the lacrimal trough and suborbital area can be obtained for at least 12 months. In particular, due to the characteristics of low elastic modulus, high viscous modulus, and high cohesion of the gel, the filled contour is extremely natural, and the gel is not prone to displacement.

Comparative Example 9:

The elastic modulus and viscous modulus at 1 Hz made of hyaluronic acid of non-animal origin are respectively 231 and 30 Pa, tanδ value is 0.13, cohesion is 1.89N, interface normal stress is 1505N/m ² , pushing force The cross-linked hyaluronic acid gel (20mg/ml) with 20N and interface normal stress/ ^{extension force of 75m -2} was put into a 1ml sterile glass syringe, and a 27G1/2 straight sharp needle was used in the tear groove and orbit. Injection is performed from the superficial layer to the middle layer of the lower dermis. Before the injection, the patient was given preoperative local anesthesia or nerve block anesthesia. Use retrograde injection or microsphere filling for injection, depending on the severity of the volume loss and the degree of zygomatic protrusion. If the bone surface is flat and the area of volume loss is extensive, needle withdrawal injection or microball injection can be used to avoid irregular contours. After completing the injection, gently massage the treatment area to keep it in line with the contours of the surrounding tissues. If necessary, apply ice temporarily to reduce redness and swelling.

By applying the cross-linked hyaluronic acid gel described in this comparative example, it has been found that when the lacrimal trough and suborbital dermis are filled for 6 months, local diffusion and collapse occur, and the plastic effect is reduced. At 12 months, the plastic effect had almost disappeared.

Example 9: Injection and filling of the gel in the lips

The elastic modulus and viscous modulus at 1 Hz made of hyaluronic acid of non-animal origin are 105 and 84 Pa, tanδ value is 0.8, cohesion force is 4.56N, interface normal stress is 3631N/m ² , pushing force The cross-linked hyaluronic acid gel (20mg/ml) with 8.3N and interface normal stress/extruding force of 437m ^-2 is put into a 1ml sterile glass syringe, and a 27G1/2 straight needle is used in the lip dermis Inject from the shallow layer to the middle layer. Before the injection, the patient was given preoperative local anesthesia or nerve block anesthesia. The injection can be started from the arch of the lips or the corners of the mouth, but the retrograde injection method must be used. The lip edge is injected first, which helps limit the eversion of the upper and lower lips. After completing the injection, gently massage the treatment area to keep it in line with the contours of the surrounding tissues. If necessary, apply ice temporarily to reduce redness and swelling.

By applying the cross-linked sodium hyaluronate gel of the present invention, it is observed that a satisfactory filling effect can be obtained on the lips for at least 6 months. In particular, due to the characteristics of low elastic modulus, high viscous modulus, and high cohesion of the gel, the filled contour is extremely natural, and the gel is not prone to displacement.

Comparative Example 9:

The elastic modulus and viscous modulus at 1 Hz made of hyaluronic acid of non-animal origin are respectively 231 and 30 Pa, tanδ value is 0.13, cohesion is 1.89N, interface normal stress is 1505N/m ² , pushing force The cross-linked hyaluronic acid gel (20mg/ml) with 20N and interface normal stress/ ^{extruding force of 75m -2} is put into a 1ml sterile glass syringe, and a 27G1/2 straight needle is used to shallow the lip dermis. Layer to middle layer for injection. Before the injection, the patient was given preoperative local anesthesia or nerve block anesthesia. The injection can be started from the arch of the lips or the corners of the mouth, but the retrograde injection method must be used. The lip edge is injected first, which helps limit the eversion of the upper and lower lips. After completing the injection, gently massage the treatment area to keep it in line with the contours of the surrounding tissues. If necessary, apply ice temporarily to reduce redness and swelling.

Through the application of the cross-linked sodium hyaluronate gel described in this comparative example, it has been found that the plasticity of the product is relatively unnatural. When the lips are filled for 3 months, partial collapse occurs and the plastic effect is reduced. At 6 months, the plasticity is reduced. The plastic effect has almost disappeared.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the embodiments, and any other changes, modifications, combinations, substitutions, and simplifications made without departing from the spirit and principle of the present invention All should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims

The preparation method of cross-linked hyaluronic acid gel is characterized in that it comprises at least the following steps:

(1) Mix the aqueous solution of hyaluronic acid and the cross-linking agent uniformly in a non-neutral environment to form a mixed solution;

(2) Put the mixed solution at a temperature lower than 0°C and higher than the eutectic point of the mixed solution to form a solid-liquid heterogeneous system;

(3) The solid-liquid heterogeneous system is placed at a temperature lower than 0° C. and higher than the eutectic point of the mixed solution for cross-linking reaction.
The method according to claim 1, further comprising the step of melting the solid phase in the solid-liquid heterogeneous system after the cross-linking reaction, and optionally further comprising the steps of neutralization, purification and homogenization.
The method according to any one of the preceding claims, wherein the solid-liquid heterogeneous system is composed of ice crystals and a hyaluronic acid solution.
The method according to any one of the preceding claims, wherein the aqueous solution of hyaluronic acid in step (1) comprises hyaluronic acid, a metal salt of hyaluronic acid, or a hyaluronic acid derivative or comprises a hyaluronic acid selected from hyaluronic acid, hyaluronic acid and hyaluronic acid. An aqueous solution of a mixture of at least two of the metal salt of sulfonic acid and the derivative of hyaluronic acid.
The method according to claim 4, wherein the metal hyaluronic acid salt is selected from the group consisting of sodium salt, potassium salt, magnesium salt, calcium salt, zinc salt and combinations thereof of hyaluronic acid.
The method according to claim 4, wherein the hyaluronic acid derivative is combined with polysaccharides containing hydroxyl groups, for example selected from carboxymethyl cellulose, alginate, chondroitin-4-sulfate, chondroitin-6 -Sulfate, xanthan gum, chitosan, pectin, agar, carrageenan, guar gum derivatives, and mixtures thereof.
The method according to any one of the preceding claims, wherein the formation temperature of the solid-liquid heterogeneous system in step (2) is the eutectic point of the mixed solution to 0°C, preferably the eutectic point of the mixed solution to -5°C, even It is the eutectic point of the mixed solution to -10°C.
The method according to any one of the preceding claims, wherein the molecular weight of hyaluronic acid used for cross-linking is between 10 and 5 million, especially between 500,000 and 4 million, and even between 500,000 and 2 million.
A cross-linked hyaluronic acid gel, wherein the elastic modulus of the gel is about 10 Pa to about 1000 Pa at 1 Hz; the viscous modulus of the gel is about 10 Pa to about 500 Pa at 1 Hz; the viscous modulus of the gel The ratio of tanδ to elastic modulus is about 0.4 to about 1.5 at 1 Hz; the cohesion is about 2N to about 10N.
The cross-linked hyaluronic acid gel according to claim 9, wherein the cross-linked hyaluronic acid gel conforms to the following formula:

300m -2 <interface normal stress / push-out force <1500m -2 .
The cross-linked hyaluronic acid gel according to any one of the preceding claims 9-10, which has at least one selected from the following:

-The tanδ value of the crosslinked hyaluronic acid gel is about 0.6 to about 1.5 at 1 Hz, preferably about 0.6 to about 1;

-The cohesive force of the cross-linked hyaluronic acid gel is about 4N to about 10N, preferably about 4N to about 8N;

-The HA concentration of the cross-linked hyaluronic acid gel is greater than 16 mg/ml, preferably 20-25 mg/ml, more preferably 21-23 mg/ml;

-The viscosity modulus of the cross-linked hyaluronic acid gel is about 10 Pa to about 500 Pa at 1 Hz, preferably 80-100 Pa;

-The elastic modulus of the cross-linked hyaluronic acid gel is 10 Pa to 300 Pa at 1 Hz, preferably 50 Pa to 200 Pa;

-The protein content of the cross-linked hyaluronic acid gel is not more than 0.1% by mass;

-The heavy metal content of the cross-linked hyaluronic acid gel is not more than 5 μg/g;

-The residual amount of the cross-linking agent of the cross-linked hyaluronic acid gel is not more than 2 μg/g; and/or

-The bacterial endotoxin content of the cross-linked hyaluronic acid gel is not more than 0.5 EU/mL.
The cross-linked hyaluronic acid gel according to any one of the preceding claims 9-11, wherein the gel comprises cross-linked hyaluronic acid cross-linked with 1,4-butanediol diglycidyl ether (BDDE) ( HA).
The cross-linked hyaluronic acid gel according to any one of the preceding claims 9-12, which is prepared by the method according to any one of the preceding claims 1-8.
A composition comprising the cross-linked hyaluronic acid gel obtained according to the method of any one of the preceding claims 1-8, or the cross-linked hyaluronic acid gel according to any one of the preceding claims 9-13 .
Medical devices such as dressings, implants, or fillings, which comprise the cross-linked hyaluronic acid gel obtained according to the method of any one of the preceding claims 1-8, and the cross-linked hyaluronic acid gel obtained according to any one of the preceding claims 9-13. The cross-linked hyaluronic acid gel, or the composition according to any one of the preceding claims 14.