CN113045779A

CN113045779A - Cross-linked hyaluronic acid gel crude product and preparation method thereof

Info

Publication number: CN113045779A
Application number: CN202110303097.3A
Authority: CN
Inventors: 吴万福; 苏江伟; 刘建建; 潘存才; 张燕; 郭学平
Original assignee: Bloomage Biotech Co Ltd
Current assignee: Bloomage Biotech Co Ltd
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2021-06-29
Anticipated expiration: 2041-03-22
Also published as: CN113045779B

Abstract

The invention provides a cross-linked hyaluronic acid gel crude product, wherein 6 points are randomly selected from the cross-linked hyaluronic acid gel crude product with the volume of less than 8 cubic decimeters, and the hyaluronic acid content of the crude product at the points is measured, wherein the relative standard deviation of 6 measurement results is less than or equal to 0.06. The present invention also provides a method for preparing a crosslinked hyaluronic acid gel, the method comprising the steps of: mixing hyaluronic acid or its salt, cross-linking agent and acidic or alkaline solution, and dissolving in solvent₁Stirring at the temperature to obtain a raw material mixture; placing the stirred raw material mixture in situ at T₂And carrying out crosslinking reaction at the temperature to obtain a crosslinked hyaluronic acid gel crude product. The hyaluronic acid gel has good mixing uniformity and crosslinking efficiencyThe product has high content of the polysaccharide, excellent enzymolysis resistance, can effectively prolong the in-vivo maintenance time, has small risk of pollution in the production process and high safety, and can be widely used in the medical and American fields as a tissue filler.

Description

Cross-linked hyaluronic acid gel crude product and preparation method thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a cross-linked hyaluronic acid gel and a preparation method thereof.

Background

Hyaluronic Acid (HA), also known as Hyaluronic acid, is a long-chain polymer composed of alternating arrangements of D-glucuronic acid and N-acetylglucosamine, HAs good hydrophilicity, viscoelasticity, lubricity and biocompatibility, and HAs been widely used in the medical field.

Because natural HA HAs the defects of poor stability, sensitivity to hyaluronidase and free radicals, short retention time in vivo, lack of mechanical strength in a water system and the like, the development and application of the natural HA in the field of biomedicine are limited. The physical and chemical properties of the HA gel can be changed through various chemical modifications and crosslinking reactions, so that the mechanical strength and the degradation resistance of the HA gel are improved, new HA crosslinking derivatives with biological activity and functionality are obtained, and novel medical biomaterials are further developed.

The current common crosslinking method mainly comprises the steps of uniformly mixing a crosslinking agent and hyaluronic acid in a solution, and carrying out a crosslinking reaction by heat preservation. The uniform mixing process of HA is generally realized by a mechanical stirring mode, such as a mechanical pressurization stirrer or a planetary stirrer, but the rotating speed of a stirring paddle of the stirring mode is generally not high, otherwise, the materials are easy to splash, and meanwhile, the materials can be subjected to a stronger shearing action, so that the materials are denatured, therefore, the uniform mixing can be realized only by stirring at a low rotating speed for a long time (the stirring time is generally more than 2 hours), and the HA raw materials can be degraded by themselves after being stirred in an acidic or alkaline solution for an overlong time, and the physical and chemical properties of the terminal products can be obviously influenced. The crosslinking process is in a static state, and the crosslinking uniformity greatly depends on the stirring uniformity. In addition, the traditional stirring mode generally needs to transfer the uniformly mixed materials into a specific container for temperature-controlled crosslinking, so that the material waste is inevitable, and the risk of material pollution is greatly increased. Therefore, to overcome the deficiencies of the prior art crosslinking methods, improvements in the mixing and crosslinking process are desired.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a cross-linked hyaluronic acid gel and a preparation method thereof.

Specifically, the present invention relates to the following aspects:

1. the cross-linked hyaluronic acid gel crude product is characterized in that 6 points are randomly selected from the cross-linked hyaluronic acid gel crude product with the volume of less than 8 cubic decimeters, and the hyaluronic acid content of the crude product at the points is measured, wherein the relative standard deviation of 6 measurement results is less than or equal to 0.06.

2. The crude hyaluronic acid gel according to item 1, wherein the average value of the hyaluronic acid content of the crude crosslinked hyaluronic acid gel measured at 6 points is 50 to 300 mg/g.

3. The crude hyaluronic acid gel according to item 1, wherein 6 points are randomly selected per 8 cubic decimeters or less of the crude crosslinked hyaluronic acid gel to measure the degree of modification of the crude hyaluronic acid gel at the point, and the relative standard deviation of the 6 measurement results is 0.06 or less.

4. The crude hyaluronic acid gel according to item 3, wherein the modification degree of the crude crosslinked hyaluronic acid gel measured at 6 points is 0.6% to 5%.

5. The crude hyaluronic acid gel according to item 1, wherein the time for complete in vitro degradation of the crosslinked crude hyaluronic acid gel is not less than 160 min.

6. A method of preparing a cross-linked hyaluronic acid gel, comprising the steps of:

mixing hyaluronic acid or its salt, cross-linking agent and acidic or alkaline solution, and dissolving in solvent₁Stirring at the temperature to obtain a raw material mixture;

placing the stirred raw material mixture in situ at T₂And carrying out crosslinking reaction at the temperature to obtain a crosslinked hyaluronic acid gel crude product.

7. The method according to item 6, wherein T is-5 ℃ or more₁At the temperature of less than or equal to 10 ℃, the stirring time is 0-30min。

8. The method of item 7, wherein T is₁-T_1-0At most 2 ℃, wherein T is_1-0The temperature at which stirring was initiated.

9. The method according to item 6, wherein the stirring is performed in such a manner that the hyaluronic acid or a salt thereof, the crosslinking agent, and the acidic or alkaline solution after mixing are revolved around a given center while being rotated around the center of a container for holding the raw materials to be mixed, wherein the stirring is performed with a revolving radius of 5 to 80cm, preferably 10 to 60cm, a revolving speed of 0 to 3500 rpm, preferably 100 to 3000 rpm, and a rotating speed of 0 to 3500 rpm, preferably 100 to 3000 rpm.

10. The method according to item 6, wherein T is 20 ℃ or more₂The temperature is less than or equal to 60 ℃, and the crosslinking reaction time is 2-60 h.

11. The method of item 10, wherein T is₂-T_2-0At most 2 ℃, wherein T is_2-0Is the temperature at which the crosslinking reaction is initiated.

12. The method according to item 6, wherein the crosslinking reaction is carried out in such a manner that the stirred raw material mixture is revolved around a given center while being rotated around the center of a container holding the raw material mixture, wherein the revolution radius at the time of the crosslinking reaction is 5 to 80cm, preferably 10 to 60cm, the revolution speed is 0 to 3500 rpm, preferably 50 to 2500 rpm, and the rotation speed is 0 to 3500 rpm, preferably 50 to 2500 rpm.

13. The method according to item 6, wherein the acidic solution has a pH of 2 to 5 and the basic solution has a pH of 9 to 13.

14. The method of item 6, wherein the crosslinking agent is one or more of diglycidyl ether, divinyl sulfone, 1,2,7, 8-diepoxyoctane, 1, 3-diepoxybutane, and sodium trimetaphosphate.

15. The method according to item 6, wherein the mass ratio of the crosslinking agent to the hyaluronic acid or a salt thereof is 0.2 to 8: 100.

17. A hyaluronic acid gel for injection, which is obtained by purifying the crude hyaluronic acid gel according to any one of items 1 to 5 or the crude hyaluronic acid gel prepared by the method according to any one of items 6 to 15.

The method can complete the material mixing time from several hours to several minutes, controls the material at a lower temperature in the stirring process, ensures the mixing uniformity of the material, and effectively avoids the degradation of the HA raw material in an acidic or alkaline solution. Compared with the traditional crosslinking, the mixing uniformity and the crosslinking uniformity of the dynamic crosslinking adopted by the method are greatly improved. The mixing and crosslinking processes of the method are carried out in the reactor, secondary transfer is not needed, the whole process does not need to be opened, and the risk of product pollution is greatly reduced. The hyaluronic acid gel disclosed by the invention is good in mixing uniformity, high in crosslinking efficiency and excellent in enzymolysis resistance, can effectively prolong the in-vivo maintenance time, is low in pollution risk of products in the production process, is high in safety, and can be widely used in the medical and aesthetic fields as a tissue filler.

Drawings

FIG. 1 is a schematic diagram of a crude gel sample according to the present invention;

FIG. 2 is a schematic view of the construction of a temperature-controlled centrifugal blender used in the present invention.

Detailed Description

The present invention is further illustrated by the following examples, which are intended to be purely exemplary of the invention and are not intended to be limiting.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in experimental or practical applications, the materials and methods are described below. In case of conflict, the present specification, including definitions, will control, and the materials, methods, and examples are illustrative only and not intended to be limiting. The present invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.

To determine the homogeneity of the crude crosslinked hyaluronic acid gel, the following methods are generally used in the art: randomly taking 6 samples from a certain volume of crude hyaluronic acid gel, measuring the hyaluronic acid content and the modification degree of the samples, and calculating the relative standard deviation of the measurement results.

The content of hyaluronic acid is the mass of hyaluronic acid contained in a unit mass of the crude crosslinked hyaluronic acid gel. For example, the content of hyaluronic acid in the crude gel is 50mg/g, which means that 50mg of hyaluronic acid is contained in each g of the crude gel. The modification degree is the degree of modification of the HA macromolecule by the cross-linking agent, and is the molar ratio of the modified disaccharide units in the chain structure of hyaluronic acid to the total disaccharide units. The Relative Standard Deviation (RSD) is also called standard deviation coefficient, variation coefficient, etc. the precision of the result can be analyzed in the inspection and detection work by dividing the standard deviation by the corresponding average value and multiplying by 100%.

In a specific embodiment, 6 random points are taken in the crude hyaluronic acid gel of the present invention per 8 cubic decimeters or less to determine the hyaluronic acid content of the crude hyaluronic acid at the points. The gel crude product with the volume of less than 8 cubic decimeters can be in any shape, such as a cylinder, a sphere, a cube and the like, the random sampling of 6 points means that 6 points can be randomly sampled from the gel crude product with the volume, and the sample amount of the 6 points can be the same or different. The relative standard deviation of the 6 measurements is ≦ 0.06, e.g., 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0. The cross-linked hyaluronic acid gel has good uniformity and uniform cross-linking reaction.

In one embodiment, the crude gel is cylindrical. As shown in FIG. 1, samples of a certain mass of gel were taken at A, B, C, D, E, F locations, respectively, of the crude gel. Wherein A and C are respectively positioned at the central positions of two bottom surfaces of the cylinder, and B is positioned at the midpoint of a connecting line of A and C. D. F, E are parallel to the central axis and are located on the side of the cylinder, where D and F are located on the top and bottom sides, respectively, and E is located at the midpoint of the DF line. A. B, C, D, E, F the sampling weight at the six points may be any weight that meets the needs of the subsequent measurement, and may be the same or different. In one specific embodiment, A, B, C, D, E, F takes 1g for each of the six points.

Further, the crude crosslinked hyaluronic acid gel measured at 6 points has an average value of hyaluronic acid content of 50 to 300mg/g, for example, 50mg/g, 60mg/g, 70mg/g, 80mg/g, 90mg/g, 100mg/g, 110mg/g, 120mg/g, 130mg/g, 140mg/g, 150mg/g, 160mg/g, 170mg/g, 180mg/g, 190mg/g, 200mg/g, 210mg/g, 220mg/g, 230mg/g, 240mg/g, 250mg/g, 260mg/g, 270mg/g, 280mg/g, 290mg/g, 300 mg/g. Wherein the average values described herein are arithmetic average values.

The hyaluronic acid content in the crude gel product can be determined by any method known in the art, such as the method of appendix E of YY/T0962-2014. In a specific embodiment, the amount of hyaluronic acid in the gel sample is determined with reference to the method of standard YY/T0962-.

In a specific embodiment, the modification degree of the crude crosslinked hyaluronic acid gel at the point is determined at random at 6 points in the crude product per 8 cubic decimeters or less, and the relative standard deviation of the 6 determination results is less than or equal to 0.06, such as 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.

The modification degree of the crude crosslinked hyaluronic acid gel measured at the 6 points is 0.6% to 5%, for example, 0.6%, 1%, 2%, 3%, 4%, 5%.

The degree of modification can be determined using methods known in the art, for example, according to area normalization, and is calculated from the peak area of the modified fragment in a size exclusion chromatogram. In one embodiment, the degree of modification is determined by reference to: measurement of degree of modification of Poplar Biddio. BDDE Cross-Linked hyaluronic acid gel the measurement method described in [ D ]. 2015. The higher the degree of modification, the more sufficient the crosslinking reaction, and the higher the crosslinking efficiency.

In a specific embodiment, the in vitro complete degradation time of the cross-linked hyaluronic acid gel is more than or equal to 160min, such as 160min, 170min, 180min, 190min, 200min, 210min, 220min, 230min, 240min, 5h, 6h, 7h, 10 h. The method for measuring the in vitro degradation time comprises the following steps: taking a proper amount of sample to be detected (about containing HA8mg), placing the sample in a penicillin bottle, adding 4mL of self-made HA enzyme (the enzyme activity is 600IU/mL), and uniformly mixing by vortex. Shaking in a water bath at 42 deg.C, sampling 50 μ L every 10min and diluting appropriately, measuring absorbance at 232nm until absorbance no longer changes, considering degradation is complete, and recording the time taken for complete degradation. The in vitro degradation has important reference significance for predicting the maintenance time of the modified sodium hyaluronate gel for injection after being injected into a human body. The longer the complete degradation time in vitro, the longer the in vivo maintenance time.

In a specific embodiment, the cross-linked hyaluronic acid gel is cross-linked by hyaluronic acid having a molecular weight of 20-200 ten thousand.

The present invention also provides a method for preparing a crosslinked hyaluronic acid gel, characterized in that it comprises the following steps:

Since hyaluronic acid, particularly high molecular weight hyaluronic acid, is relatively viscous and can be degraded in acidic or alkaline solutions, it is important to keep the reaction system at a lower temperature to avoid degradation while sufficiently stirring the reaction system.

In a specific embodiment, the temperature during stirring is such that T is-5 ℃ or less₁≤10℃，T₁For example, the temperature may be-5 deg.C, -4 deg.C, -3 deg.C, -2 deg.C, -1 deg.C, 0 deg.C, 1 deg.C, 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C, 6 deg.C, 7 deg.C, 8 deg.C, 9 deg.C, 10 deg.C, and the stirring time may be 0-30 min.

Further, T₁-T_1-0At most 2 ℃, wherein T is_1-0The temperature at which stirring was initiated. E.g. a temperature of 0 ℃ at the start of stirring, the temperature T during stirring₁Less than or equal to 2 ℃. If the temperature at the beginning of the stirring is 10 ℃, the temperature during the stirringDegree T₁≤12℃。

In a specific embodiment, the temperature during the crosslinking reaction is 20 ℃ T or less₂≤60℃，T₂Can be at 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C, 60 deg.C, and the crosslinking reaction time is 2-60 h.

Further, T₂-T_2-0At most 2 ℃, wherein T is_2-0Is the temperature at which the crosslinking reaction is initiated. For example, the temperature at the beginning of the crosslinking reaction is 40 ℃ so that the temperature T of the crosslinking reaction₁≤42℃。

Namely, the temperature needs to be controlled in the stirring process and the crosslinking reaction process, so that the change amplitude is in a smaller range, and the formation of the crosslinked hyaluronic acid gel crude product with high uniformity is facilitated.

The crosslinking reaction of the stirred mixture in the "in-situ" state means that the reaction is continued in the original reaction apparatus without transferring the stirred mixture to another reactor to carry out the reaction after the stirring of the mixture. Therefore, the mixing and crosslinking processes of the raw materials are carried out in the reactor, secondary transfer is not needed, the whole process does not need to be opened, and the risk of product pollution is greatly reduced.

The two steps of stirring and crosslinking reaction are carried out under stirring, wherein the stirring mode adopts a mode of combining revolution and rotation. Wherein, revolution refers to the circular motion of an object along a certain orbit by taking another object as the center; the trajectory along may be circular, elliptical, hyperbolic, or parabolic. Self-rotation refers to the self-rotation of an object, which rotates along an axis passing through the object. In the present invention, revolution means that the container for holding the mixed raw material is rotated along the central axis of the rotary table, and rotation means that the container for holding the mixed raw material is rotated along the central axis thereof.

In a specific embodiment, the stirring is performed in such a manner that the mixed hyaluronic acid or a salt thereof, the crosslinking agent, and the acidic or alkaline solution are revolved around a given center while being rotated around the center of the container for holding the mixed raw materials, wherein the revolution radius during the stirring is 5 to 80cm, preferably 10 to 60cm, the revolution speed is 0 to 3500 rpm, preferably 100-.

In a specific embodiment, the crosslinking reaction is carried out in such a manner that the stirred raw material mixture is revolved around a given center while being rotated around the center of a container in which the raw material mixture is placed, wherein the revolution radius at the time of the crosslinking reaction is 5 to 80cm, preferably 10 to 60cm, the revolution speed is 0 to 3500 revolutions per minute, preferably 50 to 2500 revolutions per minute, and the rotation speed is 0 to 3500 revolutions per minute, preferably 50 to 2500 revolutions per minute.

In a specific embodiment, the acidic solution has a pH of 2 to 5, and may be a hydrochloric acid solution, an acetic acid solution, or the like, and the basic solution has a pH of 9 to 13, and may be a sodium hydroxide solution, a potassium hydroxide solution, or the like.

In a specific embodiment, the crosslinking agent is one or more of diglycidyl ether, divinyl sulfone, 1,2,7, 8-diepoxyoctane, 1, 3-diepoxybutane, and sodium trimetaphosphate.

In a specific embodiment, the mass ratio of the cross-linking agent to hyaluronic acid or a salt thereof is 0.2-8: 100.

The invention also provides a hyaluronic acid gel for injection, which is obtained by further purifying the hyaluronic acid gel or the hyaluronic acid gel prepared by the method.

According to the invention, stirring and crosslinking reaction are carried out in a revolution and rotation mode, and the temperature in the stirring and crosslinking reaction process is strictly controlled, so that the material stirring time can be greatly shortened, and the degradation of the HA raw material is effectively reduced; the prepared crude hyaluronic acid gel product has good uniformity, the relative standard deviation of the content and the modification degree of hyaluronic acid in the crude hyaluronic acid gel product is less than or equal to 0.06, and the in vitro complete degradation time of the crude hyaluronic acid gel product can be greatly prolonged to more than 160 min. In addition, the method of the invention has the advantages of totally closed operation in the mixing and crosslinking processes, no need of transferring materials and uncapping operation, effective control of the microbial level of products, reduction of the risk of product pollution and guarantee of the product safety.

The following examples and comparative examples were prepared in a local hundred-level environment in a ten-thousand-level clean zone, the solutions used were all prepared with water for injection and filtered through a 0.22 μm filter membrane; all the instruments are soaked and cleaned by 2 percent sodium hydroxide solution and then sterilized by moist heat or dry heat; the materials, reagents and the like used are commercially available unless otherwise specified. Wherein the weight of the hyaluronic acid is calculated by pure product.

Examples

Example 1

The crosslinked hyaluronic acid gel for injection is prepared according to the following steps:

adding 5g hyaluronic acid with molecular weight of 200 kilodalton (Da), 0.1g 1, 4-butanediol diglycidyl ether and 25g sodium hydroxide solution with pH value of 13 into a reactor, placing the reactor in a temperature-controlled centrifugal stirrer, and setting stirring starting temperature T of the temperature-controlled centrifugal stirrer_1-0The stirring time was 30min at 0 ℃, the stirring speed (rotation/revolution) was 2000/400RPM, and the temperature at the end of stirring was 1.2 ℃. The crosslinking temperature was set at 35 ℃, the crosslinking speed (rotation/revolution) was 2000/400RPM, the crosslinking time was 20 hours, and the temperature at the end of crosslinking was 36.3 ℃. And obtaining a crude product of the crosslinked hyaluronic acid gel after the reaction is finished. Wherein, the stirring and the crosslinking reaction are both carried out in a temperature-controlled centrifugal stirrer.

The structure of the temperature-controlled centrifugal stirrer is shown in figure 1, and comprises a rotary stirring bin 1 and a motor placing bin 2, wherein a rotary platform 3, a reactor 4 and a pressure temperature control system are arranged in the rotary stirring bin 1, and a revolution motor 5 and a revolution controller 6 are arranged in the motor placing bin 2. The bottom of the rotary platform 3 is connected with an output shaft of a revolution motor 5, the reactor 4 is fixed on the rotary platform 3 through a clamp, the reactor 4 is driven by the rotary platform 3 to revolve, meanwhile, the reactor 4 can rotate, and the rotation of the reactor 4 is controlled by a rotation controller 6.

The pressure temperature control system comprises a temperature probe, a pressure temperature sensor 7, a pressure release valve 8, an electromagnetic valve 10, a temperature control medium pipeline 12, a temperature control medium nozzle 13 and a controller 9. The temperature probe is arranged in the reactor 4, the pressure and temperature sensor 7 is arranged on the inner wall of the rotary stirring bin 1, the electromagnetic valve 10 is arranged on the temperature control medium pipeline 12, the temperature control medium nozzle 13 is positioned in the rotary stirring bin 1 and communicated with the temperature control medium pipeline 12, and the pressure release valve 8 is positioned at a pressure release port of the rotary stirring bin 1; the temperature probe and the pressure temperature sensor 7 are connected to the input end of the controller 9, and the electromagnetic valve 10 and the pressure release valve 8 are connected to the output end of the controller 9.

The temperature probe is wirelessly connected with the controller 9.

The temperature control medium comprises a refrigerant and a heating medium, the refrigerant comprises liquid nitrogen and liquid carbon dioxide, and the heating medium comprises hot water, hot air or steam.

The stirring bin upper cover 11 is provided with a human-computer interaction module, and the human-computer interaction module is used for setting the working information of the stirrer.

When the temperature-controlled centrifugal stirrer is used, the materials are packaged into the reactor 4, the upper cover of the rotary stirring bin 1 is closed, the stirring time, the revolution and rotation speed and the target temperature in the reactor are set, the type of the temperature-controlled medium is selected, and stirring is started. Along with rotatory stirring starts, the material temperature slowly rises in the reactor 4, temperature probe transmits real-time temperature to controller 9, controller 9 combines accuse temperature medium classification and cooling efficiency according to the difference of actual temperature and target temperature, automatic calculation accuse temperature medium injection volume, through opening and shutting of control solenoid valve 10 with accuse temperature medium through accuse temperature medium injection nozzle 13 spray to rotatory stirring storehouse, the pressure temperature control system also can monitor the temperature in the rotatory stirring storehouse and pressure automatic control relief valve 8 opens and shuts simultaneously, with material temperature control at target temperature. The controller 9 automatically calculates the ejection volume of the temperature control medium according to the difference between the actual temperature and the target temperature, in combination with the type of the temperature control medium and the cooling efficiency, which is a conventional technical means in the field, and the description of this embodiment is omitted.

Example 2

Example 2 differs from example 1 in that the stirring time was 4min, the temperature at the end of the stirring was 0.5 ℃, and the reaction conditions were otherwise the same as in example 1.

Example 3

Example 3 differs from example 1 in that the stirring time was 10min, the temperature at the end of the stirring was 0.8 ℃, and the reaction conditions were otherwise the same as in example 1.

Example 4

Example 4 differs from example 1 in the stirring initiation temperature T_1-0The temperature was-5 ℃ and the temperature at the end of stirring was-4.0 ℃ and the reaction conditions were the same as in example 1.

Example 5

Example 5 differs from example 1 in the stirring initiation temperature T_1-0The temperature was 10 ℃ and the temperature at the end of stirring was 11.4 ℃ and the reaction conditions were the same as in example 1.

Example 6

Example 6 is different from example 1 in that the molecular weight of the raw material hyaluronic acid is 100 ten thousand Da, the sodium hydroxide solution with pH value of 13 is changed into the acetic acid solution with pH value of 2, the stirring temperature is 10 ℃, the stirring time is 10min, the crosslinking temperature is 60 ℃, and the crosslinking time is 2 h.

Example 7

Example 7 is different from example 1 in that the molecular weight of the raw material hyaluronic acid is 20 ten thousand Da, the sodium hydroxide solution with pH value of 13 is changed into the acetic acid solution with pH value of 5, the stirring temperature is 2 ℃, the stirring time is 2min, the crosslinking temperature is 20 ℃, and the crosslinking time is 60 h.

Comparative example 1

Comparative example 1 is different from example 5 in that the temperature is not controlled in the stirring step, i.e., the stirring start temperature is the same as in example 5, but the temperature is different at the end of the stirring.

Comparative example 2

Comparative example 2 is different from example 5 in that the temperature is not controlled in the crosslinking reaction step, i.e., the crosslinking reaction starting temperature is the same as in example 5, but the temperature is different at the end of the crosslinking reaction.

Comparative example 3

Comparative example 3 is different from example 5 in that the stirring initiation temperature is 15 c, and other reaction conditions are the same as example 5.

Comparative example 4

Comparative example 4 is different from example 5 in that the stirring time is 60min, and other reaction conditions are the same as example 5.

Comparative example 5

Comparative example 5 is different from example 5 in that the crosslinking reaction is not performed in situ, specifically, the raw materials are mixed and stirred in a traditional planetary stirrer, and after the stirring is finished, the stirred mixture is transferred to a water bath for the crosslinking reaction. Wherein, in the traditional planetary stirrer, the stirring time is set to be 30min, the stirring speed (rotation/revolution) is 200/50RPM, and the stirring temperature is 25 ℃ at room temperature. And after stirring, putting the reactor into a water bath at 35 ℃ for crosslinking for 20 hours to obtain a crosslinked hyaluronic acid gel crude product.

Comparative example 6

Comparative example 6 differs from example 5 in that the stirring start temperature was 10 ℃ but the temperature during stirring was not controlled and the temperature at the end of stirring was 22.6 ℃. Other reaction conditions were the same as in example 5.

The reaction conditions of the above examples and comparative examples are specifically shown in Table 1.

TABLE 1 parameters of the examples and comparative examples

And (3) performance testing:

1. hyaluronic acid feedstock base/acid degradation test

The hyaluronic acid molecule can be characterized by testing the molecular weight of the hyaluronic acid molecule, wherein the hyaluronic acid molecule can generate molecular chain breakage in an acidic or alkaline solution. After the examples and comparative examples were stirred well, 1g of sample was randomly taken. The molecular weight was determined and compared to the starting molecular weight to study the degradation rate of hyaluronic acid. The test results are shown in Table 2. The molecular weight was measured using a DAWN HELEOS II type multi-angle laser light scattering apparatus according to the instructions of the apparatus.

Table 2 molecular weight test results after hyaluronic acid stirring

2. Hyaluronic acid content test

The crude crosslinked hyaluronic acid gels prepared in the examples and the comparative examples are randomly sampled in 6 samples in the gel below 8 cubic decimeters, each sample is 1g, phosphate sodium chloride buffer solution is added to purify the gel until the gel weight is 8g, and the hyaluronic acid content of the purified 6 samples is determined by referring to the method of standard YY/T0962-2014 appendix E.

TABLE 3 sample point mixing uniformity test results

3. Initial contamination test

Samples before sterilization after filling in the examples and the comparative examples were respectively taken and tested for initial contamination according to the microorganism examination method in the appendix of the pharmacopoeia of the people's republic of China 2020 edition. The test results are shown in Table 4.

Table 4 initial contamination test results of examples and comparative examples

	Initial contaminating bacteria (cfu/mL)
		Examples1	＜10
Example 2	＜10
		Example 3	＜10
Example 4	＜10
		Example 5	＜10
Example 6	＜10
		Example 7	＜10
Comparative example 1	＜10
		Comparative example 2	＜10
Comparative example 3	＜10
		Comparative example 4	＜10
Comparative example 5	67
		Comparative example 6	＜10

4. Test for degree of modification

The modification degree was measured by the following method: measurement of degree of modification of Poplar Biddio. BDDE Cross-Linked hyaluronic acid gel the measurement method described in [ D ]. 2015. The results of the modification degree test are shown in Table 5.

5. In vitro degradation test

The in vitro degradation has important reference significance for predicting the maintenance time of the modified sodium hyaluronate gel for injection after being injected into a human body. The longer the complete degradation time in vitro, the longer the in vivo maintenance time. The in vitro degradation time of the crude crosslinked hyaluronic acid gels prepared in the thorough examples and comparative examples was determined using the in vitro degradation time determination method described herein above. The test results are shown in Table 5.

TABLE 5 examples, comparative examples the modification degrees and in vitro degradation test results

	Degree of modification (%)	Complete degradation time (min)
			Example 1	1.82	190
Example 2	1.81	190
			Example 3	1.82	190
Example 4	1.86	200
			Example 5	1.81	190
Example 6	2.62	210
			Example 7	2.38	240
Comparative example 1	1.59	150
			Comparative example 2	1.64	120
Comparative example 3	1.70	150
			Comparative example 4	1.73	160
Comparative example 5	1.03	100
			Comparative example 6	1.06	100

The results in table 2 show that the HA molecule is degraded in a strong alkaline/strong acidic environment, and the degradation is more pronounced at higher temperatures and longer times. The traditional planetary mixer needs at least 2 hours to uniformly stir the reaction system, and the HA raw material is obviously degraded in the process. The invention adopts revolution and rotation modes for stirring, can uniformly mix materials within 30min, strictly controls the temperature in the stirring process, and can effectively reduce the degradation of the HA raw material.

The results in table 3 show that the crosslinked HA gel prepared by the present invention HAs very high uniformity, and the actually measured standard deviation is about one two hundredth of that of the traditional mechanical stirring, which indicates that the dynamic crosslinking adopted by the method of the present invention can ensure that the materials are dynamically and uniformly mixed in the crosslinking reaction process, so that the crosslinking uniformity is greatly improved.

The results in table 4 show that, by using the method of the present invention, the mixing and crosslinking processes are all operated in a closed manner, the materials do not need to be transferred, and the operation of opening the cover is not needed, so that the microbial level of the product is effectively controlled, the risk of product pollution is reduced, and the safety of the product is ensured.

The results in table 5 show that, with the method of the present invention, the stirring time is short, the degradation of HA raw material is low, the crosslinking reaction proceeds to a high degree, and the modification degree of the prepared product is significantly higher than that of the conventional crosslinking manner, which indicates that the crosslinking efficiency of the method of the present invention is significantly improved, and the product with the equivalent modification degree can be obtained on the premise of ensuring a lower amount of the crosslinking agent in the process development of a new product. In addition, the in vitro degradation maintenance time of the crosslinked hyaluronic acid gel for injection prepared by the method is obviously superior to that of a comparative example, and the in vivo maintenance time of the product prepared by the method can be more than 15 months according to the in vitro complete degradation time.

In conclusion, the method greatly shortens the material stirring time, effectively reduces the degradation of the HA raw material, and improves the crosslinking efficiency and the in-vivo maintaining time. The product prepared by the method can meet the physical and chemical properties and biocompatibility required by tissue fillers and joint cavity injection products, and can be widely applied to the technical fields of medical cosmetology and biological medical treatment.

Claims

2. The crude hyaluronic acid gel according to claim 1, wherein the average value of the hyaluronic acid content of the crude crosslinked hyaluronic acid gel measured at 6 points is 50-300 mg/g.

3. The crude hyaluronic acid gel according to claim 1, wherein the degree of modification of the crude hyaluronic acid gel at the point is measured at 6 random points per 8 cubic decimeters or less of the crude hyaluronic acid gel, and the relative standard deviation of the 6 measurements is 0.06 or less.

4. The crude hyaluronic acid gel according to claim 3, wherein the modification degree of the crude crosslinked hyaluronic acid gel measured at 6 points is 0.6-5%.

5. The crude hyaluronic acid gel according to claim 1, wherein the time for complete degradation in vitro of the crude crosslinked hyaluronic acid gel is 160min or more.

7. The method according to claim 6, wherein-5 ℃ T.ltoreq.T₁The stirring time is 0-30min at the temperature of less than or equal to 10 ℃.

8. The method of claim 7, wherein T is₁-T_1-0At most 2 ℃, wherein T is_1-0The temperature at which stirring was initiated.

9. The method according to claim 6, wherein the stirring is performed in such a manner that the mixed hyaluronic acid or a salt thereof, the crosslinking agent and the acidic or alkaline solution are revolved around a given center while being rotated around the center of a container for holding the mixed raw materials, wherein the stirring is performed with a revolving radius of 5 to 80cm, preferably 10 to 60cm, a revolving speed of 0 to 3500 rpm, preferably 100-.

10. The method of claim 6, wherein T is 20 ℃ ≦ T₂The temperature is less than or equal to 60 ℃, and the crosslinking reaction time is 2-60 h.