Recombinant collagen mediated cross-linked sodium hyaluronate gel and preparation method thereof
Through the recombinant collagen-mediated crosslinking method, the problem of excessive BDDE residue in sodium hyaluronate gel was solved, which reduced the risk of adverse reactions after surgery, and enhanced the stability and retention time of the gel through collagen regeneration.
Patent Information
- Application Number
- CN202510578796.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-07
- Publication Date
- 2025-07-25
AI Technical Summary
The residual amount of crosslinking agent BDDE in existing sodium hyaluronate gels is too high, resulting in an increased risk of adverse reactions in patients after surgery and lacks the function of stimulating collagen regeneration.
Recombinant collagen-mediated crosslinking method is used to mix sodium hyaluronate with recombinant type III humanized collagen, and epoxy activator is added to form a hydrogel, and the activator not involved in the reaction is removed by chemical washing to form stable ether bond crosslinking.
Reduce crosslinking agent residues, reduce the risk of adverse reactions after surgery, and stimulate collagen regeneration, improving gel stability and retention time in the body.
Smart Images
Figure CN120361305A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of sodium hyaluronate gels, and in particular, to a sodium hyaluronate gel crosslinked by recombinant collagen and a preparation method thereof. Background Art
[0002] Sodium hyaluronate (hyaluronan, abbreviated as HA) is a natural polysaccharide widely distributed in the cytoplasm and intercellular matrix of the body, playing a lubricating and nourishing role for the cells and cell organs contained therein. HA molecules form an interactive network structure through non-covalent interactions, with the chains winding around each other to form a complex network structure. The HA molecular chain contains a large number of hydroxyl groups, which can adsorb hundreds of times its own weight of water, achieving the effect of moisturizing. However, due to the relatively loose structure between the overall HA molecules, it is easily degraded and damaged, so the functions such as moisturizing are maintained for a short time, which greatly limits the application of HA in facial filling.
[0003] Research has found that the loose structure of HA molecules can be strengthened by crosslinking. After crosslinking, the physical support of the HA gel is significantly improved, the stability of the HA gel is enhanced, and the retention time of HA in the body can be extended. The crosslinking mechanism of HA molecules: the groups on the crosslinking agent are activated and covalently connected to the free carboxyl or hydroxyl groups on the HA molecules to form ether bonds. This stable chemical bond transforms the HA molecules from the original loose and disordered state into a complex and stable connection network. After the free carboxyl and hydroxyl groups on the HA molecules are combined, the cleavage efficiency of the specific recognition cleavage site by hyaluronidase decreases, thereby extending the retention time of the crosslinked sodium hyaluronate gel in the body.
[0004] Currently, the crosslinking agents used in commercially available crosslinked sodium hyaluronate products are mostly 1,4-butanediol diglycidyl ether (BDDE). The epoxy groups on BDDE are activated and react with the hydroxyl groups on HA to form ether bonds, thereby obtaining crosslinked sodium hyaluronate products. As a commonly used crosslinking agent, BDDE is an epoxy compound with high cytotoxicity. It is difficult to metabolize in the human body, and too high a residue is likely to cause various postoperative adverse reactions and increase the risk of infection for patients. Therefore, for crosslinked sodium hyaluronate gel products, controlling the residue amount of BDDE is a particularly crucial issue. Finding a new HA crosslinking method, while not affecting its physical support performance, to improve the production process and reduce the usage amount of BDDE in sodium hyaluronate gels is particularly important. In addition, adding non-toxic and absorbable collagen to the gel to supplement and stimulate the regeneration of its own collagen on the basis of filling is also a new development direction of current market products. Summary of the Invention
[0005] The object of the present invention is to propose a new cross-linking method for sodium hyaluronate to solve the problem of excessive residual amount of BDDE in the cross-linking agent in the commercially available cross-linked sodium hyaluronate gel. In addition, it can increase the effect of stimulating collagen regeneration and reduce the occurrence of postoperative adverse symptoms in patients during the sodium hyaluronate gel filling surgery to a greater extent.
[0006] The present invention provides a method for preparing a sodium hyaluronate gel cross-linked by recombinant collagen, which comprises the following steps: 1) Sodium hyaluronate and recombinant type III humanized collagen are fully mixed and contacted to form a sodium hyaluronate-recombinant type III humanized collagen composite system; 2) An epoxy group activator is added to the sodium hyaluronate-recombinant type III humanized collagen composite system to strengthen cross-linking to form a hydrogel; 3) Using a chemical washing process, the unreacted epoxy group activator in the cross-linked gel is removed to obtain a sodium hyaluronate gel product cross-linked by recombinant collagen.
[0007] The technical solution of the present invention has the following advantages compared with the prior art: First, sodium hyaluronate (HA) and recombinant type III humanized collagen are mixed. The free amino group of collagen and the carboxyl group of sodium hyaluronate can cross-link through non-covalent interactions. Then, an epoxy group activator is added to the system. The epoxy functional group of the activator will undergo a ring-opening reaction and form a stable ether bond with the hydroxyl group of sodium hyaluronate. The cross-linking reaction forms a hydrogel. Finally, the unreacted epoxy group activator is removed by chemical washing, and the hydrogel forms a gel particle product. This preparation method successfully reduces the residue of epoxy compounds in the gel particle product and greatly reduces the occurrence of postoperative adverse symptoms in patients filled with sodium hyaluronate gel. By cross-linking recombinant type III humanized collagen, collagen regeneration can also be achieved, and the regenerated collagen is non-toxic, easy to absorb, can be used as a matrix to supplement the collagen of the filled organism, and plays a role in lubrication and nourishment, further reducing the occurrence of postoperative adverse symptoms in patients.
[0008] In a possible implementation manner, the mixing ratio of recombinant type III humanized collagen to sodium hyaluronate in step 1) is 10-30% (w / w).
[0009] In a possible implementation manner, the molecular weight of sodium hyaluronate is 1200000-1500000 Da, and the molecular weight of recombinant type III humanized collagen is 45000-65000 Da.
[0010] Mixing recombinant type III humanized collagen and sodium hyaluronate with the above mass percentage content and the preferred molecular weight, with moderate molecular weights and appropriate ratios, can obtain a more suitable mixed product - a composite system.
[0011] In a possible implementation, the activator in step 2) is one or more of 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, and epichlorohydrin.
[0012] In a possible implementation, the molar ratio of the epoxy group activator to sodium hyaluronate added is 0.2 - 0.5%.
[0013] The above-mentioned activators, 1,4-butanediol diglycidyl ether (abbreviated as BDDE), ethylene glycol diglycidyl ether (abbreviated as EGDE), or epichlorohydrin (abbreviated as ECH), all contain epoxy groups, and the epoxy groups can hydrolyze and open the ring to participate in the reaction during the cross-linking process. Moreover, the above several activators are relatively common activators, and the materials are convenient to obtain.
[0014] In a possible implementation, the cross-linking temperature in step 2) is 30 - 50 °C; the cross-linking time is 2 - 4 h.
[0015] The optimized cross-linking temperature can facilitate the opening of the epoxy groups in the activator, and the optimized cross-linking time facilitates the formation of ether bonds with HA after opening the ring and cross-linking together, making the entire recombinant collagen-mediated cross-linking more complete.
[0016] In a possible implementation, the chemical washing process in step 3) is to first wash the product after strengthening cross-linking in step 2) 4 - 8 times in an ethanolamine solution, and then wash the ethanolamine-washed product 4 - 8 times with physiological saline.
[0017] In a possible implementation, the concentration of the ethanolamine solution is 0.1 - 0.5% (v / v).
[0018] In the above scheme, because the activators used are several common simple ether substances, washing with an ethanolamine solution can cause a nucleophilic reaction between the residual epoxy groups on the activator and the primary amino groups of ethanolamine. The epoxy group opens, and N replaces one terminal oxygen, thus changing from an ether compound to an alcohol that can dissolve in physiological saline. Then, washing with physiological saline removes the residual ethanolamine component, making the obtained sodium hyaluronate gel product milder and suitable for use by patients.
[0019] In a possible implementation, it further includes a step of homogenizing the sodium hyaluronate gel product obtained in step 3). The homogenizing treatment is to form a gel particle product with uniform particle size. A colloid mill or a crusher can be used for the homogenizing treatment.
[0020] The present invention also provides a sodium hyaluronate gel cross-linked by recombinant collagen, which is prepared by the preparation method described above.
[0021] In a possible implementation, the content of sodium hyaluronate in the sodium hyaluronate gel is 2.0 - 2.7% (w / w).
[0022] In a possible implementation, the content of recombinant type III humanized collagen in the sodium hyaluronate gel is 0.2 - 0.8% (w / w).
[0023] In the above solution, sodium hyaluronate, as an important component of the sodium hyaluronate gel, always has a higher content than the recombinant collagen that mediates implantation to maintain the basic properties of the gel. The intervention of an appropriate content of recombinant type III humanized collagen can, on the one hand, play a good role in mediating crosslinking, and on the other hand, play a role in increasing the stimulation of collagen regeneration.
[0024] In a possible implementation, the degradation products of the sodium hyaluronate gel include amino acids, glucuronic acid, and N-acetylglucosamine.
[0025] In the above technical solution, the degradation products of the collagen-mediated crosslinked sodium hyaluronate gel obtained are small molecule simple amino acids, aldehydic acids, and glucosamines. Such products have safe decomposition products after being implanted into patients and are easily absorbed, degraded, and metabolized by the human body quickly.
[0026] The beneficial effects of the present invention are as follows: The present invention uses recombinant type III humanized collagen as a mediating substance to mediate and crosslink sodium hyaluronate. First, recombinant type III humanized collagen forms a non-covalent bond crosslink with sodium hyaluronate to reduce the free carboxyl groups on sodium hyaluronate. Then, an activator containing an epoxy group is further introduced to crosslink the hydroxyl groups of sodium hyaluronate to form a reinforced crosslink and form a hydrogel, so that a stable ether bond is formed between the hydrogel and the activator during crosslinking. This mediating crosslinking method can reduce the dosage of the activator, but at the same time does not reduce the chemical bond crosslinking stability of the sodium hyaluronate gel product, can extend the retention time of the crosslinked sodium hyaluronate gel in the body, and at the same time greatly reduce the occurrence of postoperative adverse symptoms in patients undergoing facial filling. This method is highly operable, simple, and cost-saving. Description of the Drawings
[0027] Figure 1 It is a physical diagram of different forms during the preparation process of the sodium hyaluronate gel described in the present invention: where Figure 1 (A) is the form after the materials are mixed evenly and crosslinked in a water bath; Figure 1 (B) is the product form after the crosslinked gel is washed with physiological saline; Figure 1 (C) is the form after the product after washing is crushed by a colloid mill; Figure 1 (D) is the form after the crushed gel is centrifuged to remove air bubbles. Detailed Embodiments
[0028] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following provides a detailed description of specific embodiments of the present invention. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable variations derived therefrom are still within the protection scope of the claims of the present invention.
[0029] It should be noted that the endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.
[0030] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In some cases, for the purpose of clarification or facilitation of citation, terms with conventional understood meanings are defined herein, and such definitions herein should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments is carried out according to the protocols and parameters given by the manufacturers. Example 1
[0031] 1) Sodium hyaluronate with a molecular weight of 1,400,000 Da and recombinant type III humanized collagen with a molecular weight of 50,000 Da were used. The recombinant type III humanized collagen and sodium hyaluronate were fully mixed at a ratio of 10%, 15%, 20%, 25%, 30% (w / w) to form a composite system.
[0032] 2) The activator BDDE with a molar ratio of 0.3% to sodium hyaluronate was poured into the well-mixed composite system and stirred evenly.
[0033] 3) The well-mixed system was placed in a 40 °C water bath for a cross-linking reaction for 4 h.
[0034] 4) The gel after cross-linking was placed in a stirring tank at 100 rpm / min and washed thoroughly with an ethanolamine solution with a concentration of 0.1% (v / v) to remove the unreacted BDDE during the cross-linking process.
[0035] 5) Then the gel was washed with physiological saline and repeatedly washed 8 times in a stirring tank at 100 rpm / min to remove the residual ethanolamine in the gel.
[0036] 6) The gel after cleaning is homogenized using a colloid mill to obtain cross-linked sodium hyaluronate gel.
[0037] The content of sodium hyaluronate in the gel is detected by high performance liquid chromatography, and the detection results are shown in Table 1.
[0038] Table 1 Effects of mixing recombinant type III humanized collagen and sodium hyaluronate at different mass ratios on the contents of sodium hyaluronate and recombinant type III humanized collagen in the final gel The mass ratio of recombinant type III humanized collagen to sodium hyaluronate, with the activator added being BDDE Sodium hyaluronate content in the gel (mg / g) Recombinant type III humanized collagen content in the gel (mg / g) Gel crosslinking degree 10% 23.5 2.4 0.37% 15% 22.3 3.3 0.35% 20% 20.4 4.1 0.31% 25% 17.7 4.5 0.25% 30% 12.9 3.5 0.15% After testing, the contents of sodium hyaluronate, collagen and cross-linking degree in the gel are shown in Table 1. When the activator is BDDE, as the mass ratio of recombinant type III humanized collagen to sodium hyaluronate increases, the content of recombinant type III humanized collagen in the gel first increases and then decreases, and the contents of sodium hyaluronate and cross-linking degree in the gel both decrease to varying degrees. Because when the proportion of recombinant type III humanized collagen in the total is appropriate, cross-linking can be mediated with less cross-linking agent. However, when the proportion of recombinant type III humanized collagen in the total is too large, the cross-linking sites of sodium hyaluronate will be blocked by collagen, resulting in a loose texture of the cross-linked gel, and the support filling effect and maintenance time are both greatly reduced. Therefore, the proportion of recombinant type III humanized collagen should be moderate. The addition of recombinant type III humanized collagen with an appropriate proportion ensures a high content of sodium hyaluronate in the product, while reducing the addition amount of BDDE activator. In this example, the addition amount of BDDE only needs to be 0.3%. As is well known, BDDE is a cross-linking agent, and its residual amount corresponds to the cross-linking degree in the product. It can be seen from the data in the above table that the cross-linking degree corresponding to the BDDE residual amount in this example can reach below 0.37%, while the cross-linking degree of gel products on the market is generally above 1%, indicating that the BDDE residual amount in this example is small. The high content of recombinant type III humanized collagen in the product indicates that the product has the effect of collagen regeneration. Example 2
[0039] 1) Sodium hyaluronate with a molecular weight of 1400000 Da and recombinant type III humanized collagen with a molecular weight of 50000 Da are used, and recombinant type III humanized collagen and sodium hyaluronate are fully mixed at a ratio of 10%, 15%, 20%, 25%, 30% (w / w) to form a composite system.
[0040] 2) The activator EGDE with a molar ratio of 0.3% to sodium hyaluronate is poured into the mixed composite system and stirred evenly.
[0041] 3) The fully mixed system is placed in a water bath at 40 °C for cross-linking reaction for 4 h.
[0042] 4) The gel after crosslinking is placed in a stirring tank at 100 rpm / min and thoroughly washed with a 0.1% (v / v) ethanolamine solution to remove the unreacted EGDE during the crosslinking process.
[0043] 5) Then the gel is washed with physiological saline and repeatedly washed 8 times in a stirring tank at 100 rpm / min to remove the residual ethanolamine in the gel.
[0044] 6) The washed gel is homogenized using a colloid mill to obtain a crosslinked sodium hyaluronate gel.
[0045] The content of sodium hyaluronate in the gel is detected by high performance liquid method, and the detection results are shown in Table 2.
[0046] Table 2 Effects of mixing recombinant type III humanized collagen and sodium hyaluronate at different mass ratios on the contents of sodium hyaluronate and recombinant type III humanized collagen in the final gel The mass ratio of recombinant type III humanized collagen to sodium hyaluronate, with the activator added being EGDE Sodium hyaluronate content in the gel (mg / g) Recombinant type III humanized collagen content in the gel (mg / g) Gel crosslinking degree 10% 22.3 2.3 0.35% 15% 21.5 3.2 0.32% 20% 19.2 3.9 0.27% 25% 16.4 4.2 0.21% 30% 12.7 3.5 0.16% After testing, the contents of sodium hyaluronate, collagen and crosslinking degree in the gel are shown in Table 2. When the activator is EGDE, as the mass ratio of recombinant type III humanized collagen to sodium hyaluronate increases, the content of recombinant type III humanized collagen in the gel first increases and then decreases, the content of sodium hyaluronate and crosslinking degree in the gel decrease, and the texture of the crosslinked gel is loose, and the support filling effect and maintenance time are reduced. In this example, the crosslinking degree of the product is below 0.35%, corresponding to a small residual amount of the activator EGDE. The high content of recombinant type III humanized collagen in the product indicates that the product has the effect of collagen regeneration. Example 3
[0047] 1) Sodium hyaluronate with a molecular weight of 1400000 Da and recombinant type III humanized collagen with a molecular weight of 50000 Da are used, and recombinant type III humanized collagen and sodium hyaluronate are fully mixed at a ratio of 10%, 15%, 20%, 25%, 30% (w / w) to form a composite system.
[0048] 2) The activator ECH with a molar ratio of 0.3% to sodium hyaluronate is poured into the mixed composite system and stirred evenly.
[0049] 3) The fully mixed system is placed in a water bath at 40 °C for crosslinking reaction for 4 h.
[0050] 4) The gel after crosslinking is placed in a stirring tank at 100 rpm / min and thoroughly washed with a 0.1% (v / v) ethanolamine solution to remove the unreacted ECH during the crosslinking process.
[0051] 5) The gel was then washed with saline and repeated 8 times in a stirring tank at 100 rpm / min to remove the remaining ethanolamine in the gel.
[0052] 6) The cleaned gel is homogenized using a colloid mill to obtain a cross-linked sodium hyaluronate gel.
[0053] The sodium hyaluronate content in the gel was detected by high performance liquid chromatography (HPLC). The test results are shown in Table 3.
[0054] Table 3 Effect of mixing recombinant humanized type III collagen and sodium hyaluronate at different mass ratios on the content of sodium hyaluronate in the final gel The mass ratio of recombinant type III humanized collagen to sodium hyaluronate, with the activator added being ECH Sodium hyaluronate content in the gel (mg / g) Recombinant type III humanized collagen content in the gel (mg / g) Gel crosslinking degree 10% 21.7 2.2 0.34% 15% 20.3 3.0 0.31% 20% 18.4 3.7 0.26% 25% 15.2 3.8 0.22% 30% 12.6 3.5 0.15% After testing, the sodium hyaluronate, collagen content and cross-linking degree in the gel are shown in Table 3. When the activator is ECH, the mass ratio of recombinant type III humanized collagen to sodium hyaluronate increases, the content of recombinant type III humanized collagen in the gel first increases and then decreases, the sodium hyaluronate content and cross-linking degree in the gel decrease, the cross-linked gel texture is loose, and the supporting filling effect and maintenance time are reduced. And using different types of cross-linking agents, the properties of the gel will also have different degrees of difference. In this embodiment, the cross-linking degree of the product is below 0.34%, corresponding to the small amount of activator ECH residue. The content of recombinant type III humanized collagen in the product is high, indicating that the product has produced the effect of collagen regeneration. Example 4
[0055] 1) Sodium hyaluronate with a molecular weight of 1,400,000 Da and recombinant humanized type III collagen with a molecular weight of 50,000 Da were used, and the recombinant humanized type III collagen and sodium hyaluronate were fully mixed at a ratio of 10% (w / w) to form a composite system.
[0056] 2) Pour the activator BDDE in a molar ratio of 0.2%, 0.3%, 0.4%, and 0.5% to sodium hyaluronate into the mixed composite system and stir evenly.
[0057] 3) Place the thoroughly mixed system in a 40°C water bath for 4 hours of cross-linking reaction.
[0058] 4) After cross-linking, the gel was placed in a stirring tank at 100 rpm / min and thoroughly washed with 0.1% (v / v) ethanolamine solution to remove the BDDE that did not participate in the reaction during the cross-linking process.
[0059] 5) The gel was then washed with saline and repeated 8 times in a stirring tank at 100 rpm / min to remove the remaining ethanolamine in the gel.
[0060] 6) The cleaned gel is homogenized using a colloid mill to obtain a cross-linked sodium hyaluronate gel.
[0061] The content of sodium hyaluronate in the gel was detected by high performance liquid chromatography, and the test results are shown in Table 4.
[0062] Table 4 Effects of adding BDDE and sodium hyaluronate at different molar ratios on the content of sodium hyaluronate in the gel Molar ratio of BDDE to sodium hyaluronate Sodium hyaluronate content in the gel (mg / g) Recombinant type III humanized collagen content in the gel (mg / g) Gel crosslinking degree 0.2% 20.1 2.0 0.27% 0.3% 23.5 2.3 0.37% 0.4% 25.6 2.6 0.43% 0.5% 28.3 2.8 0.56% After testing, the content of sodium hyaluronate, collagen and crosslinking degree in the gel are shown in Table 4. When the activator is BDDE, as the molar ratio of the activator to sodium hyaluronate increases, the content of recombinant type III humanized collagen, sodium hyaluronate and crosslinking degree in the gel also increase. The crosslinked gel is more supportive, and the support filling effect and maintenance time are improved. In this example, the crosslinking degree of the product is below 0.56%, corresponding to a small residual amount of the activator BDDE. The high content of recombinant type III humanized collagen in the product indicates that the product has the effect of collagen regeneration. Example 5
[0063] 1) Sodium hyaluronate with a molecular weight of 1400000 Da and recombinant type III humanized collagen with a molecular weight of 50000 Da were used. The recombinant type III humanized collagen and sodium hyaluronate were fully mixed at a ratio of 10% (w / w) to form a composite system.
[0064] 2) Activator EGDE with a molar ratio of 0.2%, 0.3%, 0.4%, 0.5% to sodium hyaluronate was poured into the mixed composite system and stirred evenly.
[0065] 3) The fully mixed system was placed in a water bath at 40 °C for crosslinking reaction for 4 h.
[0066] 4) The gel after crosslinking was placed in a stirring tank at 100 rpm and washed thoroughly with 0.1% (v / v) ethanolamine solution to remove the unreacted EGDE during the crosslinking process.
[0067] 5) Then the gel was washed with normal saline, and the washing was repeated 8 times in a stirring tank at 100 rpm to remove the residual ethanolamine in the gel.
[0068] 6) The washed gel was homogenized using a colloid mill to obtain a crosslinked sodium hyaluronate gel.
[0069] The content of sodium hyaluronate in the gel was detected by high performance liquid chromatography, and the test results are shown in Table 5.
[0070] Table 5 Effects of adding EGDE and sodium hyaluronate at different molar ratios on the content of sodium hyaluronate in the gel Molar ratio of EGDE to sodium hyaluronate Sodium hyaluronate content in the gel (mg / g) Recombinant type III humanized collagen content in the gel (mg / g) Gel crosslinking degree 0.2% 19.3 2.0 0.26% 0.3% 22.3 2.2 0.35% 0.4% 24.3 2.4 0.44% 0.5% 27.7 2.8 0.55% After testing, the contents of sodium hyaluronate, collagen and the crosslinking degree in the gel are shown in Table 5. When the activator is EGDE, as the molar ratio of the activator to sodium hyaluronate increases, the contents of recombinant humanized type III collagen, sodium hyaluronate and the crosslinking degree in the gel also increase. The crosslinked gel has better support, and the support filling effect and the maintenance time are improved. In this example, the crosslinking degree of the product is below 0.55%, corresponding to a small residual amount of the activator EGDE. The high content of recombinant humanized type III collagen in the product indicates that the product has the effect of collagen regeneration. Example 6
[0071] 1) Sodium hyaluronate with a molecular weight of 1400000 Da and recombinant humanized type III collagen with a molecular weight of 50000 Da are used. The recombinant humanized type III collagen and sodium hyaluronate are fully mixed at a ratio of 10% (w / w) to form a composite system.
[0072] 2) Activator ECH with a molar ratio to sodium hyaluronate of 0.2%, 0.3%, 0.4%, 0.5% is poured into the mixed composite system and stirred evenly.
[0073] 3) The fully mixed system is placed in a water bath at 40 °C for a crosslinking reaction for 4 h.
[0074] 4) The gel after crosslinking is placed in a stirring tank at 100 rpm / min and washed thoroughly with a 0.1% (v / v) ethanolamine solution to remove the unreacted ECH during the crosslinking process.
[0075] 5) Then the gel is washed with physiological saline and repeatedly washed 8 times in a stirring tank at 100 rpm / min to remove the residual ethanolamine in the gel.
[0076] 6) The washed gel is homogenized using a colloid mill to obtain a crosslinked sodium hyaluronate gel.
[0077] The content of sodium hyaluronate in the gel is detected by high performance liquid chromatography, and the detection results are shown in Table 6.
[0078] Table 6 Effects of adding ECH and sodium hyaluronate at different molar ratios on the content of sodium hyaluronate in the gel Molar ratio of ECH to sodium hyaluronate Sodium hyaluronate content in the gel (mg / g) Recombinant type III humanized collagen content in the gel (mg / g) Gel crosslinking degree 0.2% 21.7 1.7 0.23% 0.3% 20.3 2.1 0.34% 0.4% 22.4 2.2 0.42% 0.5% 25.8 2.5 0.51% After testing, the contents of sodium hyaluronate, collagen and the crosslinking degree in the gel are shown in Table 6. When the activator is ECH, as the molar ratio of the activator to sodium hyaluronate increases, the contents of recombinant humanized type III collagen, sodium hyaluronate and the crosslinking degree in the gel also increase. The crosslinked gel is more supportive, and the support filling effect and the maintenance time are improved. Moreover, when using different types of crosslinking agents, the properties of the gel will also have different degrees of differences. In this example, the crosslinking degree of the product is below 0.51%, corresponding to a small residual amount of the activator ECH. The content of recombinant humanized type III collagen in the product is high, indicating that the product has the effect of collagen regeneration. Example 7
[0079] 1) Sodium hyaluronate with a molecular weight of 1,400,000 Da and recombinant humanized type III collagen with a molecular weight of 50,000 Da are used. The recombinant humanized type III collagen and sodium hyaluronate are fully mixed at a ratio of 10% (w / w) to form a composite system.
[0080] 2) The activator BDDE with a molar ratio of 0.3% to sodium hyaluronate is poured into the mixed composite system and stirred evenly.
[0081] 3) The fully mixed system is placed in a water bath at 30, 37, 40, 45, 50 °C for a crosslinking reaction for 4 h.
[0082] 4) The gel after crosslinking is placed in a stirring tank at 100 rpm and washed thoroughly with a 0.1% (v / v) ethanolamine solution to remove the unreacted BDDE during the crosslinking process.
[0083] 5) Then the gel is washed with normal saline, and the washing is repeated 8 times in a stirring tank at 100 rpm to remove the residual ethanolamine in the gel.
[0084] 6) The washed gel is homogenized using a colloid mill to obtain a crosslinked sodium hyaluronate gel.
[0085] The content of BDDE (reflecting the crosslinking degree) in the crosslinked gel is detected by high performance liquid chromatography, and the detection results are shown in Table 7.
[0086] Table 7 Effects of different crosslinking temperatures on the crosslinking degree of the gel Different crosslinking temperatures, with the activator added being BDDE Gel crosslinking degree Sodium hyaluronate content in the gel (mg / g) 30 ℃ 0.15% 17.2 37 ℃ 0.25% 19.3 40 ℃ 0.37% 23.5 45 ℃ 0.42% 24.7 50 ℃ 0.45% 25.6 After testing, the sodium hyaluronate and crosslinking degree in the gel are shown in Table 7. When the activator is BDDE, with the increase of crosslinking temperature, the sodium hyaluronate content and crosslinking degree in the gel also increase. This shows that under the same conditions, high temperature can improve the crosslinking rate of the gel and accelerate the reaction. In this example, the crosslinking degree of the product is below 0.45%, corresponding to a small residual amount of the activator BDDE. The content of recombinant type III humanized collagen in the product is high, indicating that the product has the effect of collagen regeneration. Example 8
[0087] 1) Sodium hyaluronate with a molecular weight of 1,400,000 Da and recombinant type III humanized collagen with a molecular weight of 50,000 Da were used. The recombinant type III humanized collagen and sodium hyaluronate were fully mixed at a ratio of 10% (w / w) to form a composite system.
[0088] 2) The activator EGDE with a molar ratio of 0.3% to sodium hyaluronate was poured into the mixed composite system and stirred evenly.
[0089] 3) The fully mixed system was placed in a water bath at 30, 37, 40, 45, and 50 °C for 4 h of crosslinking reaction.
[0090] 4) The gel after crosslinking was placed in a stirring tank at 100 rpm and washed thoroughly with a 0.1% (v / v) ethanolamine solution to remove the unreacted EGDE during the crosslinking process.
[0091] 5) Then the gel was washed with physiological saline, and the washing was repeated 8 times in a stirring tank at 100 rpm to remove the residual ethanolamine in the gel.
[0092] 6) The washed gel was homogenized using a colloid mill to obtain a crosslinked sodium hyaluronate gel.
[0093] The content of EGDE (reflecting the crosslinking degree) in the crosslinked gel was detected by high-performance liquid chromatography, and the detection results are shown in Table 8.
[0094] Table 8 Effect of different crosslinking temperatures on the crosslinking degree of the gel Different crosslinking temperatures, with the activator added being EGDE Gel crosslinking degree Sodium hyaluronate content in the gel (mg / g) 30℃ 0.14% 17.6 37℃ 0.21% 20.2 40℃ 0.33% 22.3 45℃ 0.39% 24.3 50℃ 0.43% 25.3 After testing, the sodium hyaluronate and crosslinking degree in the gel are shown in Table 8. When the activator is EGDE, with the increase of crosslinking temperature, the sodium hyaluronate content and crosslinking degree in the gel also increase. This shows that under the same conditions, high temperature can improve the crosslinking rate of the gel and accelerate the reaction. In this example, the crosslinking degree of the product is below 0.43%, corresponding to a small residual amount of the activator EGDE. The content of recombinant type III humanized collagen in the product is high, indicating that the product has the effect of collagen regeneration. Example 9
[0095] 1) Use sodium hyaluronate with a molecular weight of 1,400,000 Da and recombinant humanized type III collagen with a molecular weight of 50,000 Da. The recombinant humanized type III collagen and sodium hyaluronate are fully mixed at a ratio of 10% (w / w) to form a composite system.
[0096] 2) Pour activator ECH with a molar ratio of 0.3% to sodium hyaluronate into the well-mixed composite system and stir evenly.
[0097] 3) Place the fully mixed system in a water bath at 30, 37, 40, 45, and 50 °C for 4 h of crosslinking reaction.
[0098] 4) The gel after crosslinking is placed in a stirring tank at 100 rpm / min and washed thoroughly with a 0.1% (v / v) ethanolamine solution to remove the unreacted ECH during the crosslinking process.
[0099] 5) Then wash the gel with normal saline and repeat the washing 8 times in a stirring tank at 100 rpm / min to remove the residual ethanolamine in the gel.
[0100] 6) The washed gel is homogenized using a colloid mill to obtain crosslinked sodium hyaluronate gel.
[0101] The content of ECH (reflecting the degree of crosslinking) in the crosslinked gel is detected by headspace gas chromatography, and the detection results are shown in Table 9.
[0102] Table 9 Effects of different crosslinking temperatures on the crosslinking degree of the gel Different crosslinking temperatures, with the activator added being ECH Gel crosslinking degree Sodium hyaluronate content in the gel (mg / g) 30 ℃ 0.11% 16.5 37 ℃ 0.17% 19.3 40 ℃ 0.25% 21.7 45 ℃ 0.34% 23.9 50 ℃ 0.39% 24.8 After testing, the content of sodium hyaluronate and the degree of crosslinking in the gel are shown in Table 9. When the activator is ECH, with the increase of the crosslinking temperature, the content of sodium hyaluronate and the degree of crosslinking in the gel also increase. It shows that under the same conditions, high temperature can improve the crosslinking rate of the gel and accelerate the reaction. And when using different types of crosslinking agents, the properties of the gel will also have different degrees of differences. In this example, the crosslinking degree of the product is below 0.39%, corresponding to a small residual amount of activator ECH. The content of recombinant humanized type III collagen in the product is high, indicating that the product has the effect of collagen regeneration. Example 10
[0103] 1) Use sodium hyaluronate with a molecular weight of 1,400,000 Da and recombinant humanized type III collagen with a molecular weight of 50,000 Da. The recombinant humanized type III collagen and sodium hyaluronate are fully mixed at a ratio of 10% (w / w) to form a composite system.
[0104] 2) Pour activator BDDE with a molar ratio of 0.3% to sodium hyaluronate into the well-mixed composite system and stir evenly.
[0105] 3) Place the well - mixed system in a 40 °C water bath for cross - linking reactions for 2, 2.5, 3, 3.5, and 4 h.
[0106] 4) Place the gel after cross - linking in a stirring tank at 100 rpm and wash it thoroughly with a 0.1% (v / v) ethanolamine solution to remove the BDDE that did not participate in the reaction during cross - linking.
[0107] 5) Then wash the gel with normal saline and repeat the washing 8 times in a stirring tank at 100 rpm to remove the residual ethanolamine in the gel.
[0108] 6) Homogenize the washed gel using a colloid mill to obtain cross - linked sodium hyaluronate gel.
[0109] Detect the content of BDDE (reflecting the cross - linking degree) in the cross - linked gel by high - performance liquid method, and the detection results are shown in Table 10.
[0110] Table 10 Effects of different cross - linking times on the cross - linking degree of the gel Different crosslinking times, with the activator added being BDDE Gel crosslinking degree Sodium hyaluronate content in the gel (mg / g) 2.0 h 0.19% 13.2 2.5 h 0.23% 16.4 3.0 h 0.29% 20.5 3.5 h 0.36% 22.4 4.0 h 0.37% 22.7 After testing, the content of sodium hyaluronate and cross - linking degree in the gel are shown in Table 10. When the activator is BDDE, as the cross - linking time increases, the content of sodium hyaluronate and cross - linking degree in the gel also increase. When the cross - linking time is about 4 h, the reaction is basically completed. At this time, the low cross - linking degree of the gel also means less cross - linking residue, which further reduces the probability of side reactions caused by the gel after injection. In this example, the cross - linking degree of the product is below 0.37%, corresponding to less residue of the activator BDDE. The high content of recombinant type III humanized collagen in the product indicates that the product has the effect of collagen regeneration. Example 11
[0111] 1) Use sodium hyaluronate with a molecular weight of 1400000 Da and recombinant type III humanized collagen with a molecular weight of 50000 Da. The recombinant type III humanized collagen and sodium hyaluronate are thoroughly mixed at a ratio of 10% (w / w) to form a composite system.
[0112] 2) Pour the activator EGDE with a molar ratio of 0.3% to sodium hyaluronate into the mixed composite system and stir evenly.
[0113] 3) Place the well - mixed system in a 40 °C water bath for cross - linking reactions for 2, 2.5, 3, 3.5, and 4 h.
[0114] 4) The gel after crosslinking is placed in a stirring tank at 100 rpm / min and thoroughly washed with a 0.1% (v / v) ethanolamine solution to remove the unreacted EGDE during the crosslinking process.
[0115] 5) Then the gel is washed with physiological saline and repeatedly washed 8 times in a stirring tank at 100 rpm / min to remove the residual ethanolamine in the gel.
[0116] 6) The washed gel is homogenized using a colloid mill to obtain crosslinked sodium hyaluronate gel.
[0117] The content of EGDE (reflecting the degree of crosslinking) in the crosslinked gel is detected by high performance liquid method, and the detection results are shown in Table 11.
[0118] Table 11 Effects of different crosslinking times on the crosslinking degree of the gel Different crosslinking times, with the activator added being EGDE Gel crosslinking degree Sodium hyaluronate content in the gel (mg / g) 2.0h 0.17% 13.1 2.5h 0.20% 15.9 3.0h 0.25% 18.2 3.5h 0.31% 22.1 4.0h 0.33% 22.3 After testing, the content of sodium hyaluronate and the degree of crosslinking in the gel are shown in Table 11. When the activator is EGDE, with the increase of the crosslinking time, the content of sodium hyaluronate and the degree of crosslinking in the gel also increase. When the crosslinking time is about 4 h, the reaction is basically completed. In this example, the crosslinking degree of the product is below 0.33%, corresponding to a small residual amount of the activator EGDE. The content of recombinant type III humanized collagen in the product is high, indicating that the product has the effect of collagen regeneration. Example 12
[0119] 1) Sodium hyaluronate with a molecular weight of 1400000 Da and recombinant type III humanized collagen with a molecular weight of 50000 Da are used. The recombinant type III humanized collagen and sodium hyaluronate are thoroughly mixed at a ratio of 10% (w / w) to form a composite system.
[0120] 2) The activator ECH with a molar ratio of 0.3% to sodium hyaluronate is poured into the mixed composite system and stirred evenly.
[0121] 3) The thoroughly mixed system is placed in a water bath at 40 °C for crosslinking reaction for 2, 2.5, 3, 3.5, 4 h.
[0122] 4) The gel after crosslinking is placed in a stirring tank at 100 rpm / min and thoroughly washed with a 0.1% (v / v) ethanolamine solution to remove the unreacted ECH during the crosslinking process.
[0123] 5) Then the gel is washed with physiological saline and repeatedly washed 8 times in a stirring tank at 100 rpm / min to remove the residual ethanolamine in the gel.
[0124] 6) The gel after cleaning is homogenized using a colloid mill to obtain cross-linked sodium hyaluronate gel.
[0125] The content of ECH (reflecting the degree of cross-linking) in the cross-linked gel is detected by headspace gas chromatography, and the detection results are shown in Table 12.
[0126] Table 12 Effects of different cross-linking times on the cross-linking degree of the gel Different crosslinking times, with the activator added being ECH Gel crosslinking degree Sodium hyaluronate content in the gel (mg / g) 2.0h 0.12% 12.9 2.5h 0.16% 14.8 3.0h 0.19% 17.3 3.5h 0.23% 21.3 4.0h 0.25% 21.7 After testing, the content of sodium hyaluronate and the cross-linking degree in the gel are shown in Table 12. When the activator is ECH, as the cross-linking time increases, the content of sodium hyaluronate and the cross-linking degree in the gel also increase. When the cross-linking time is about 4 h, the reaction is basically completed. Moreover, when using different types of cross-linking agents, the properties of the gel will also have different degrees of differences. In this example, the cross-linking degree of the product is below 0.25%, corresponding to a small residual amount of the activator ECH. The content of recombinant type III humanized collagen in the product is high, indicating that the product has the effect of collagen regeneration.
[0127] The physical diagrams of different forms during the preparation process of the sodium hyaluronate gel prepared in this application are as Figure 1 shown. As can be seen from Figure 1 (A), the gel after cross-linking in a water bath after the materials are mixed evenly presents a light yellow crystalline shape; as can be seen from Figure 1 (B), the cross-linked gel presents a colorless crystalline shape after being washed with physiological saline; as can be seen from Figure 1 (C), the product after the cleaning is completed is ground by a colloid mill and presents a colorless gel shape with uniform texture; as can be seen from Figure 1 (D), the ground gel presents a colorless transparent liquid after centrifugation to remove air bubbles.
[0128] The above is only the specific implementation manner of this application, but the protection scope of this application is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed in this application should be covered by the protection scope of this application. Therefore, the protection scope of this application should be subject to the protection scope of the claims.
Claims
1. A method for preparing a sodium hyaluronate gel crosslinked by recombinant collagen, characterized in that, It includes the following steps: Sodium hyaluronate and recombinant humanized type III collagen are fully mixed and contacted to form a sodium hyaluronate-recombinant humanized type III collagen composite system; An epoxy group activator is added to the sodium hyaluronate-recombinant humanized type III collagen composite system to strengthen crosslinking to form a hydrogel; Using a chemical washing process, the unreacted epoxy group activator in the hydrogel is removed to obtain a sodium hyaluronate gel product crosslinked by recombinant collagen.
2. The preparation method of the sodium hyaluronate gel mediated by recombinant collagen crosslinking according to claim 1, wherein In step 1), the mixing ratio of the added recombinant humanized type III collagen to sodium hyaluronate is 10-30% (w / w); Preferably, the molecular weight of sodium hyaluronate is 1,200,000-1,500,000 Da, and the molecular weight of recombinant humanized type III collagen is 45,000-65,000 Da.
3. The preparation method of the sodium hyaluronate gel mediated by recombinant collagen crosslinking according to claim 1, characterized in that, In step 2), the epoxy group activator is one or more of 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, and epichlorohydrin; Preferably, the molar ratio of the added epoxy group activator to sodium hyaluronate is 0.2-0.5%.
4. The preparation method of the sodium hyaluronate gel cross-linked by recombinant collagen according to claim 1, characterized in that, The temperature for strengthening crosslinking in step 2) is 30-50 °C; the time for strengthening crosslinking is 2-4 h.
5. The preparation method of the sodium hyaluronate gel crosslinked by recombinant collagen according to claim 1, wherein The chemical washing process in step 3) is to first wash the product after strengthening crosslinking in step 2) 4-8 times in an ethanolamine solution, and then wash the ethanolamine-washed product 4-8 times with physiological saline; Preferably, the concentration of the ethanolamine solution is 0.1-0.5% (v / v).
6. The preparation method of the sodium hyaluronate gel mediated by recombinant collagen crosslinking according to claim 1, characterized in that It also includes a step of homogenizing the sodium hyaluronate gel product obtained in step 3).
7. A sodium hyaluronate gel crosslinked by recombinant collagen, characterized in that, Prepared by the preparation method described in any one of claims 1-6.
8. The sodium hyaluronate gel crosslinked by recombinant collagen according to claim 7, wherein, The content ratio of sodium hyaluronate in the sodium hyaluronate gel is 2.0-2.7% (w / w).
9. The sodium hyaluronate gel crosslinked by recombinant collagen according to claim 7, characterized in that, The content ratio of recombinant humanized type III collagen in the sodium hyaluronate gel is 0.2-0.8% (w / w).
10. The sodium hyaluronate gel crosslinked by recombinant collagen according to claim 7, wherein, The degradation products of the sodium hyaluronate gel include amino acids, glucuronic acid, and N-acetylglucosamine.