Method for large-scale preparation of methacrylated chondroitin sulfate
By optimizing the synthesis process, including dissolution, dropping addition, crosslinking and dialysis purification steps, the problems of harsh reaction conditions and low product yields when synthesizing chondroitin sulfate in the prior art are solved, and high-efficiency and high-purity large-scale production is achieved, reducing production costs.
Patent Information
- Application Number
- CN202510236238.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-05-02
AI Technical Summary
When synthesis of chondroitin sulfate (ChSMA) in the prior art, the reaction conditions are harsh and the product yield is low, making it difficult to achieve large-scale production.
By optimizing the synthesis process, it includes dissolving chondroitin sulfate in a buffer solution, adding methacrylic anhydride dropwise at a constant speed, and cross-linking reaction under an alkaline environment, then terminating the reaction and dialysis purification to obtain high purity ChSMA.
It significantly improves the synthesis efficiency and product purity of ChSMA, solves the problem of large-scale production, reduces production costs, and simplifies the production process.
Smart Images
Figure CN119912598A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of biomaterials, and in particular to a method for large-scale preparation of methacrylylated chondroitin sulfate. Background Art
[0002] Chondroitin sulfate (ChS) is an important glycosaminoglycan widely distributed in animal connective tissues, especially in cartilage, bone, skin, cornea and blood vessel walls. Due to its unique biological functions, such as promoting bone and cartilage formation, regulating cell growth factor activity, and promoting wound healing, chondroitin sulfate is widely used in the pharmaceutical, health care and cosmetic industries. Methacryloyl chondroitin sulfate (ChSMA) is a derivative of chondroitin sulfate. By introducing methacryloyl groups into the chondroitin sulfate molecular chain, it has the characteristics of photocrosslinking. This modified ChSMA can be quickly cured under ultraviolet or visible light to form a hydrogel with good biocompatibility, so it shows great application potential in the fields of tissue engineering, drug sustained release, and biological 3D printing.
[0003] Although ChSMA has shown broad application prospects in the field of biomaterials due to its unique physical and chemical properties and biocompatibility, the demand for ChSMA is growing with the deepening of biomedical research. In particular, in the fields of three-dimensional cell culture, tissue engineering repair and personalized medicine, ChSMA has become a hot spot for research and application due to its excellent biocompatibility and plasticity.
[0004] In the prior art, the synthesis process of methacrylylated chondroitin sulfate (ChSMA) has certain deficiencies. For example, some prior art requires complex reaction conditions and long reaction time during the synthesis process, which not only increases the production cost, but also limits the feasibility of its large-scale production. In addition, the existing synthesis method is not fine enough in the regulation of reaction conditions, which makes it difficult to further improve the purity and yield of the product. These limitations make the prior art have certain challenges in meeting the market demand for high-quality, high-purity ChSMA. Summary of the invention
[0005] The present invention provides a method for large-scale preparation of methacrylylated chondroitin sulfate (ChSMA). Compared with the prior art, the present invention significantly improves the synthesis efficiency and product purity of ChSMA by optimizing the synthesis process, and solves the limitations of the traditional method such as harsh reaction conditions, low product yield and difficulty in large-scale production.
[0006] The purpose of the present invention can be achieved by the following technical solutions:
[0007] The present invention provides a method for large-scale production of methacrylylated chondroitin sulfate, comprising the following steps:
[0008] Dissolving chondroitin sulfate in a buffer solution to prepare a uniform chondroitin sulfate solution;
[0009] Add methacrylic anhydride (MA) dropwise to the chondroitin sulfate solution at a uniform rate;
[0010] After the addition of methacrylic anhydride is completed, the pH of the solution is adjusted to alkaline to achieve a cross-linking reaction between chondroitin sulfate and methacrylic anhydride to generate methacrylic acid-modified chondroitin sulfate;
[0011] After the reaction is completed, a buffer solution is added to terminate the reaction and dilute the product;
[0012] The solution after the reaction is dialyzed to remove unreacted methacrylic anhydride and its byproducts, and then freeze-dried to obtain methacrylylated chondroitin sulfate.
[0013] In one embodiment of the present invention, the buffer solution is selected as PBS solution or deionized water.
[0014] In one embodiment of the present invention, during the process of dissolving chondroitin sulfate in the buffer solution, stirring is continued until the chondroitin sulfate is completely dissolved, so that the chondroitin sulfate solution is transparent and uniform.
[0015] In one embodiment of the present invention, the concentration of chondroitin sulfate in the chondroitin sulfate solution is 40 to 50 mg / mL.
[0016] In one embodiment of the present invention, in the process of uniformly dropping methacrylic anhydride into the chondroitin sulfate solution, the dropping speed is 0.5-1 mL / min.
[0017] In one embodiment of the present invention, during the process of uniformly dropping methacrylic anhydride into the chondroitin sulfate solution, continuous stirring is maintained to ensure uniform distribution and adequate mixing of the reactants to avoid excessive local concentration or precipitation.
[0018] In one embodiment of the present invention, the amount of methacrylic anhydride added is 2% to 3% of the chondroitin sulfate solution.
[0019] In one embodiment of the present invention, after the methacrylic anhydride is added dropwise at a uniform rate, the pH of the solution is adjusted to 10-11.
[0020] In one embodiment of the present invention, the solution used to adjust the pH of the solution to alkaline is a sodium hydroxide (NaOH) solution.
[0021] In one embodiment of the present invention, the reaction time of the chondroitin sulfate and methacrylic anhydride is 3 to 5 hours to ensure sufficient reaction.
[0022] In one embodiment of the present invention, after the reaction is completed, the reaction is terminated by adding at least an equal volume of buffer solution and continuing stirring for 30 to 40 minutes to terminate the reaction and dilute the product to reduce the viscosity of the solution.
[0023] In one embodiment of the present invention, during the dialysis process, the molecular weight cutoff of the regenerated cellulose dialysis bag is 12-15 kDa to remove unreacted methacrylic anhydride and by-products.
[0024] In one embodiment of the present invention, during the dialysis process, the dialysate is replaced regularly and it is ensured that there is no more pungent odor in the solution, indicating that the dialysis process has been completed, and finally a purified methacryloyl chondroitin sulfate solution is obtained.
[0025] In one embodiment of the present invention, the dialysate is deionized water or pure water.
[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0027] The present invention significantly improves the synthesis efficiency and product purity by optimizing the reaction conditions and process in the synthesis process of methacrylylated chondroitin sulfate (ChSMA). The present invention promotes the efficient reaction of chondroitin sulfate and methacrylic anhydride by accurately controlling the pH value of the reaction system, thereby significantly improving the reaction rate and product yield. In addition, the present invention optimizes key parameters such as reaction time and dripping rate, further ensuring the uniformity of the reaction and the stability of the product. After the reaction is completed, an appropriate termination method is adopted, combined with an effective purification step, to remove unreacted by-products, significantly improving the purity of the product. This innovative process not only simplifies the production process and reduces production costs, but also provides technical support for the large-scale industrial production of ChSMA, making its application in the biomedical field more advantageous. BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 This is a schematic diagram of the reaction principle for large-scale preparation of methacrylylated chondroitin sulfate in Example 1;
[0029] Figure 2 This is the nuclear magnetic resonance spectrum of the methacryloylated chondroitin sulfate prepared in Example 1. DETAILED DESCRIPTION
[0030] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the following embodiments and / or drawings are only used to illustrate the present invention rather than to limit the present invention.
[0031] The invention provides a method for synthesizing methacrylylated chondroitin sulfate (ChSMA), which realizes an effective reaction between chondroitin sulfate and methacrylic anhydride by optimizing reaction conditions and pH adjustment. The method is carried out at room temperature, which reduces energy consumption, and improves the purity of the product through subsequent termination and dialysis steps.
[0032] In some embodiments, the amount of methacrylic anhydride added is 2% to 3% of the chondroitin sulfate solution.
[0033] In some embodiments, the methacrylic anhydride (MA) is added at a rate of 0.5 to 1 mL / min, and stirring is continued.
[0034] In some embodiments, the pH value is adjusted to 10-11 by using a NaOH solution.
[0035] In some embodiments, the termination reaction is performed by adding at least an equal volume of PBS or deionized water and diluting the product.
[0036] The following is an exemplary description of the method for preparing methacrylylated chondroitin sulfate on a large scale provided by the present invention.
[0037] Example 1
[0038] Provides a method for large-scale preparation of methacrylylated chondroitin sulfate, and a schematic diagram of the reaction principle for large-scale preparation of methacrylylated chondroitin sulfate Figure 1 , the method mainly includes the following steps.
[0039] (1) At room temperature, add 8 g of chondroitin sulfate (ChS) into 200 mL of PBS and stir until it is completely dissolved to form a uniform solution.
[0040] (2) By precisely controlling the flow rate, 4 mL of methacrylic anhydride (MA) was uniformly added to the above solution at a rate of 0.5 mL / min. During the addition, continuous stirring was maintained to ensure that the reactants were evenly distributed and fully mixed to avoid excessive local concentration or precipitation.
[0041] (3) After the addition is completed, the pH value of the system is adjusted to 10 with sodium hydroxide (NaOH) solution to create an alkaline environment to promote the smooth progress of the methacrylation reaction. In this step, the NaOH solution needs to be added slowly and the pH value is monitored in real time to ensure that it is accurately controlled within the target range.
[0042] (4) Keep the solution stirred continuously at room temperature and allow the reaction to proceed for 3 hours. During this process, the reaction conditions must be kept stable to fully realize the cross-linking reaction between chondroitin sulfate and methacrylic anhydride, thereby generating methacrylic acid-modified chondroitin sulfate.
[0043] (5) After the reaction is completed, 300 mL of PBS is added to the system and stirring is continued for 30 min to terminate the reaction and dilute the product to reduce the viscosity of the solution.
[0044] (6) The reaction solution is placed in a regenerated cellulose dialysis bag with a molecular weight cutoff of 12 kDa and dialyzed to remove unreacted methacrylic anhydride and by-products. The dialysate is replaced regularly during the dialysis process until there is no pungent odor in the dialysate, indicating that the dialysis process has been completed. After freeze-drying, purified methacryloyl chondroitin sulfate is obtained.
[0045] Figure 1 This is a schematic diagram of the reaction principle of methacrylylated chondroitin sulfate prepared in Example 1. As can be seen from the figure, the synthesis of ChSMA is through the esterification reaction of chondroitin sulfate (ChS) and methacrylic anhydride (MA), and the methacryloyl group is introduced into the ChS molecule to give it photo-crosslinking properties.
[0046] Figure 2 This is the NMR spectrum of the methacrylylated chondroitin sulfate prepared in Example 1. As can be seen from the figure, compared with ChS, the spectrum of ChSMA has new peaks around 6ppm, while these characteristic peaks are missing in the spectrum of ChS, and no obvious impurity peaks appear, which indicates that the methacrylylation reaction is successful and effectively guarantees the purity of the synthesized ChSMA.
[0047] The present invention achieves efficient and high-purity production by optimizing the synthesis process of methacrylylated chondroitin sulfate ChSMA. During the reaction process, the reaction efficiency of chondroitin sulfate and methacrylic anhydride is significantly improved by accurately controlling key parameters, thereby improving the yield of the product. At the same time, the present invention finely adjusts the reaction conditions to ensure the uniformity and stability of the reaction, thereby ensuring the consistency of the product.
[0048] The innovation of the present invention in the field of ChSMA synthesis not only solves the problems of high production cost and low efficiency in the prior art, but also broadens its potential in biomedical applications by improving the purity of the product. The proposal of this method is of great significance for promoting the wide application of ChSMA in the fields of tissue engineering, drug delivery and regenerative medicine. By realizing the large-scale production of ChSMA, the present invention helps to reduce the cost of related medical products and improve the accessibility of patients, thus having a far-reaching impact in the field of biomedicine.
[0049] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.
Claims
1. A method for large-scale preparation of methacrylylated chondroitin sulfate, characterized in that: The following steps are involved: Dissolving chondroitin sulfate in a buffer solution to prepare a uniform chondroitin sulfate solution; Add methacrylic anhydride to the chondroitin sulfate solution dropwise at a uniform rate; After the addition of methacrylic anhydride is completed, the pH of the solution is adjusted to alkaline to achieve a cross-linking reaction between chondroitin sulfate and methacrylic anhydride to generate methacrylic acid-modified chondroitin sulfate; After the reaction is completed, a buffer solution is added to terminate the reaction and dilute the product; The solution after the reaction is dialyzed to remove unreacted methacrylic anhydride and its byproducts, and then freeze-dried to obtain methacrylylated chondroitin sulfate.
2. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: The concentration of chondroitin sulfate in the chondroitin sulfate solution is 40 to 50 mg / mL.
3. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: In the process of uniformly adding methacrylic anhydride to the chondroitin sulfate solution, the dropping speed is 0.5 to 1 mL / min.
4. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: The amount of methacrylic anhydride added is 2% to 3% of the chondroitin sulfate solution.
5. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: After the methacrylic anhydride was added dropwise at a uniform rate, the pH of the solution was adjusted to 10-11.
6. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: The reaction time of the chondroitin sulfate and methacrylic anhydride is 3 to 5 hours to ensure sufficient reaction.
7. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: After the reaction is completed, the reaction is terminated by adding at least an equal volume of buffer and continuing stirring for 30 to 40 minutes to terminate the reaction and dilute the product to reduce the viscosity of the solution.
8. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: During the dialysis process, the molecular weight cutoff of the regenerated cellulose dialysis bag is 12-15 kDa to remove unreacted methacrylic anhydride and by-products.
9. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: During the dialysis process, the dialysate is replaced regularly and it is ensured that there is no more pungent odor in the solution, indicating that the dialysis process is complete and a purified methacryloylated chondroitin sulfate solution is finally obtained.
10. The method for large-scale preparation of methacryloylated chondroitin sulfate according to claim 1, characterized in that: Deionized water or purified water is selected as the dialysate.