Preparation method of chlorhexidine hyaluronate with bacteriostatic effect

Abstract

The invention discloses a preparation method of chlorhexidine hyaluronate with a bacteriostatic effect. The preparation method comprises the following steps: S1) preparing a sodium hyaluronate solution with a mass fraction of 2-3%; s2) preparing a chlorhexidine digluconate solution with the mass fraction of 1 to 2 percent; s3) slowly dropwise adding the chlorhexidine digluconate solution in the step S2 into the sodium hyaluronate solution in the step S1, and stirring for reaction; s4) adding absolute ethyl alcohol which is precooled to 4 DEG C into the reaction mixed solution, and standing and precipitating; s5) filtering and collecting a precipitate by adopting a filter membrane, and repeatedly washing for 3-5 times by sequentially using ethanol solutions with the volume ratios of 75%, 85% and 95%; and S6) drying the obtained precipitate for 8-12 hours to obtain the chlorhexidine hyaluronate. Specific process parameters and reaction conditions are adopted, the reaction temperature, the pH value and the ionic strength are accurately controlled, the synergistic effect of mechanical stirring and magnetic stirring is combined, the uniformity and controllability of the reaction are remarkably improved, and the grafting rate of the chlorhexidine hyaluronate product prepared through the method can reach 85% or above.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and specifically to a preparation method of chlorhexidine hyaluronate with antibacterial effect. Background Art

[0002] Chlorhexidine hyaluronate is a new type of antibacterial material, which is prepared by chemically modifying hyaluronic acid and chlorhexidine. Chlorhexidine hyaluronate combines the biocompatibility of hyaluronic acid and the broad-spectrum antibacterial characteristics of chlorhexidine, and has broad application prospects in the fields of medical devices, tissue engineering, drug delivery, etc. At present, it is mainly prepared by reacting sodium hyaluronate with chlorhexidine digluconate. This method has the advantages of easy availability of raw materials and mild reaction conditions. In the prior art, common preparation methods include chemical cross-linking method, physical adsorption method, electrostatic binding method, etc. Among them, the chemical cross-linking method is widely used because of its good binding stability and excellent product performance. In addition, research shows that chlorhexidine hyaluronate has more persistent antibacterial activity and lower cytotoxicity than pure chlorhexidine.

[0003] However, the following key problems exist in the prior art: (1) Temperature and pH control problems: Due to the use of a simple heating device and intermittent pH detection in the traditional process, precise temperature control and real-time pH monitoring cannot be achieved. When the temperature fluctuation of the reaction system exceeds ±2°C, the hyaluronic acid chain segments will break; when the pH value deviates from the neutral range (pH 6.8 - 7.2), the grafting efficiency of chlorhexidine will be affected. These factors ultimately lead to an overly wide molecular weight distribution range of the product (PDI > 2.0), affecting the product performance.

[0004] (2) Defects in the purification process: Currently, the method of single precipitation with 95% ethanol is commonly used for purification in industrial production. Although this method is simple to operate, since the solubility of free chlorhexidine in high-concentration ethanol is relatively low, it is difficult to completely remove it. At the same time, hyaluronic acid is prone to aggregation in high-concentration ethanol, resulting in poor purification effect of the product, and the residual amount of free chlorhexidine is usually above 2%.

[0005] (3) Insufficient control in the drying process: Existing drying processes mostly use constant temperature drying at a fixed temperature (such as 45°C), lacking temperature gradient control. Due to the thermosensitivity of chlorhexidine hyaluronate, if the temperature rises too fast in the initial stage of drying, local overheating and degradation of the product will occur; if the vacuum degree is not properly controlled in the later stage, internal moisture residues in the product will be caused, leading to agglomeration. These problems seriously affect the solubility and stability of the product.

[0006] (4) Imperfect quality evaluation system: The existing technology mainly relies on simple indicators such as product appearance and solubility for quality evaluation, lacking systematic detection methods for key parameters such as molecular weight distribution, grafting rate, and free monomer content. This results in the inability to promptly discover and solve product quality problems, causing large differences between batches and making it difficult to ensure product quality. According to statistics, for products produced using traditional methods, the relative standard deviation of the grafting rate between batches can reach more than 15%.

[0007] Therefore, there is an urgent need to develop a method for preparing chlorhexidine hyaluronate that can precisely control reaction conditions, optimize the purification process, and improve the quality stability of the product. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for preparing chlorhexidine hyaluronate to solve the problems existing in the existing method for preparing chlorhexidine hyaluronate as mentioned in the above background technology.

[0009] To achieve the above purpose, the present invention provides the following technical solution. A method for preparing chlorhexidine hyaluronate with antibacterial effect includes the following steps: S1) Dissolve sodium hyaluronate in deionized water, stir and dissolve at 40 - 45 °C for 2 - 3 hours to prepare a sodium hyaluronate solution with a mass fraction of 2 - 3%, and then cool it to room temperature; S2) Dissolve chlorhexidine digluconate in deionized water, and ultrasonically disperse it for 15 - 20 minutes to prepare a chlorhexidine digluconate solution with a mass fraction of 1 - 2%; S3) At 30 - 35 °C, slowly drop the chlorhexidine digluconate solution in step S2 into the sodium hyaluronate solution in step S1 at a dropping rate of 2 - 3 mL / min, while maintaining a stirring rate of 600 - 800 rpm. After the dropping is completed, continue to stir and react for 2 - 4 hours; S4) Slowly add pre-cooled absolute ethanol at 4 °C to the reaction mixture at a adding rate of 5 - 8 mL / min, and let it stand for precipitation for 4 - 6 hours; S5) Filter and collect the precipitate using a filter membrane with a pore size of 0.45 μm, and wash it repeatedly 3 - 5 times with ethanol solutions with a volume ratio of 75%, 85%, and 95% respectively, with each washing time not less than 30 minutes; S6) Place the obtained precipitate in a vacuum drying oven at 40 - 45 °C, heat it at a heating rate of 1 °C / min, and dry it for 8 - 12 hours, with the vacuum degree maintained at -0.08 MPa to -0.1 MPa to obtain chlorhexidine hyaluronate.

[0010] Preferably, in step S1, the molecular weight of sodium hyaluronate is 1000 - 1500 kDa, the residual protein content of sodium hyaluronate is less than 0.1%, and the heavy metal residue content is less than 10 ppm.

[0011] Preferably, when preparing the sodium hyaluronate solution in step S1, one of the following stirring methods is adopted: Mechanical stirring with a rotation speed of 200 - 400 rpm; or Magnetic stirring with a rotation speed of 300 - 500 rpm; or Combined use of mechanical stirring and magnetic stirring, where the mechanical stirring rate is 200 - 400 rpm and the magnetic stirring rate is 300 - 500 rpm.

[0012] Preferably, the ultrasonic dispersion conditions in step S2 are: Ultrasonic power: 300 - 400 W; Ultrasonic frequency: 20 - 25 kHz; Working mode: intermittent ultrasound, and the ratio of the working time to the intermittent time is 3:1.

[0013] Preferably, the specific conditions for the reaction in step S3 are: Reaction temperature: 32 ± 0.5 °C; pH value: 6.8 - 7.2, adjusted by a buffer solution; Ionic strength of the reaction system: 0.1 - 0.15 mol / L; Reaction environment: under nitrogen protection.

[0014] Preferably, in step S4: The dosage of anhydrous ethanol is 2 - 3 times the volume of the reaction mixture; The precipitation process is carried out in a low-temperature environment of 4 ± 1 °C; The precipitation process is carried out under light protection.

[0015] Preferably, the washing process in step S5 includes: S51) First, wash with 75% ethanol solution twice, 30 minutes each time; S52) Second, wash with 85% ethanol solution twice, 45 minutes each time; S53) Third, wash with 95% ethanol solution once for 60 minutes; The washing process is carried out under low-temperature and light protection conditions.

[0016] Preferably, the specific requirements for the drying process in step S6 are: S61) Heating stage: heat from room temperature to 40 - 45 °C at a rate of 1 °C / min; S62) Constant temperature stage: maintain at 40 - 45 °C for 8 - 10 hours; S63) Cooling stage: naturally cool to room temperature; S64) Vacuum degree: It gradually increases with the temperature and finally maintains at -0.08 MPa to -0.1 MPa.

[0017] Preferably, after the step S6, the following measurement steps are further included: S71) Use gel permeation chromatography to measure the molecular weight distribution and grafting rate of the product; S72) Use high performance liquid chromatography to measure the content of free chlorhexidine; S73) If the measurement results meet the following conditions, it is a qualified product: the molecular weight distribution is 800 - 1200 kDa, the grafting rate ≥ 85%, and the content of free chlorhexidine ≤ 0.5%; S74) If the above conditions are not met, the product is redissolved and purified through a ultrafiltration membrane with a cut-off molecular weight of 100 kDa. After collecting the retentate, repeat steps S4 to S6.

[0018] Compared with the prior art, the beneficial effects of the present invention are: 1) This application adopts specific process parameters and reaction conditions. By precisely controlling the reaction temperature, pH value, and ionic strength, and combining the synergistic effects of mechanical stirring and magnetic stirring, the uniformity and controllability of the reaction are significantly improved. Experimental results show that the grafting rate of the prepared chlorhexidine hyaluronic acid product can reach more than 85%, the molecular weight distribution is uniform (polydispersity index PDI ≤ 1.5), and the content of free chlorhexidine is less than 0.5%. Compared with the prior art, the present invention significantly improves the uniformity and purity of the product, laying a foundation for subsequent applications. By regulating the ionic strength of the reaction system and nitrogen protection, the degradation of the product is effectively prevented, and the reaction yield is improved. Experimental data show that under the condition of the same raw material dosage, the product yield of the present invention is increased by 15 - 20% compared with the existing method; 2) This application adopts an innovative gradient ethanol solution purification and precisely controlled temperature drying process, effectively solving the key problems in the product purification and drying processes. Through step-by-step purification with 75%, 85%, and 95% ethanol, combined with low-temperature and light-proof conditions, not only the product purity is significantly improved, but also the structural integrity of the product is maintained to the greatest extent. Compared with the traditional single-concentration ethanol purification, the gradient purification method of the present invention can increase the product purity by 10 - 15%, while effectively reducing the usage amount of organic solvents. By combining programmed heating and vacuum drying, gentle drying of the product is achieved, effectively preventing agglomeration and degradation of the product during drying. Thermogravimetric analysis results show that the product prepared by the present invention has excellent thermal stability, and the content degradation rate does not exceed 5% after storage at 60 °C for 30 days; 3) This application has established a perfect product quality control system. The products are comprehensively characterized by advanced analytical methods such as gel permeation chromatography and high performance liquid chromatography, and clear quality control indicators are set. When the product quality does not meet the standard, it can be reprocessed by ultrafiltration purification to ensure the stable and controllable quality of the final product. This quality control system not only ensures the consistency of product quality, but also significantly improves production efficiency. Experiments have proved that this method has good reproducibility between batches, with a relative standard deviation (RSD) of less than 3%. The product performance is stable and fully meets the quality requirements of the pharmaceutical grade. The process route of the present invention is simple, the operation is convenient, the equipment investment is small, and the raw material cost is low, which is suitable for industrial production. By using the method of the present invention, the large-scale production of chlorhexidine hyaluronate can be realized, providing a reliable process guarantee for its application in the fields of medical devices, biomaterials, etc.; 4) The chlorhexidine hyaluronate prepared in this application also exhibits excellent biological properties. In vitro antibacterial experiments show that the minimum inhibitory concentrations (MIC) of this product against Staphylococcus aureus and Escherichia coli are 4 μg / mL and 8 μg / mL respectively, which are significantly lower than those of unmodified chlorhexidine. The results of cytotoxicity experiments show that the half inhibitory concentration (IC50) of the product against human fibroblasts is higher than 200 μg / mL, indicating good biocompatibility. These excellent biological properties lay a foundation for the application of the product in the fields of wound dressings, anti-infection materials, etc. Description of the Drawings

[0019] Figure 1 It is a flow chart of the preparation method of this application. Detailed Embodiments

[0020] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

[0021] In the description of the invention, it should be noted that the execution order of the steps is not limited by the serial numbers. The order change of some steps, the synchronous execution of steps, the split execution of steps, etc. are all within the protection scope of this application.

[0022] Please refer to Figure 1 , the present invention provides a technical solution: a preparation method of chlorhexidine hyaluronate, including the following steps: S1) Dissolve sodium hyaluronate in deionized water, stir and dissolve at 40 - 45 °C for 2 - 3 hours to prepare a sodium hyaluronate solution with a mass fraction of 2 - 3%, and then cool it to room temperature; S2) Dissolve chlorhexidine digluconate in deionized water and ultrasonically disperse it for 15 - 20 minutes to prepare a chlorhexidine digluconate solution with a mass fraction of 1 - 2%; S3) Under the condition of 30 - 35 °C, slowly drip the chlorhexidine digluconate solution in step S2 into the sodium hyaluronate solution in step S1 at a dripping rate of 2 - 3 mL / min, while maintaining a stirring rate of 600 - 800 rpm. After the dripping is completed, continue stirring and reacting for 2 - 4 hours; S4) Slowly add anhydrous ethanol pre-cooled to 4 °C to the reaction mixture at a adding rate of 5 - 8 mL / min, and let it stand for precipitation for 4 - 6 hours; S5) Filter and collect the precipitate with a filter membrane with a pore size of 0.45 μm, and wash it repeatedly 3 - 5 times with ethanol solutions with a volume ratio of 75%, 85%, and 95% respectively, and each washing time is not less than 30 minutes; S6) Place the obtained precipitate in a vacuum drying oven at 40 - 45 °C, heat it at a heating rate of 1 °C / min, and dry it for 8 - 12 hours. The vacuum degree is maintained at -0.08 MPa to -0.1 MPa to obtain sodium hyaluronate chlorhexidine.

[0023] Specifically, by precisely controlling the process parameters of each preparation step from S1 to S6, the overall product quality has been improved.

[0024] Specifically, in step S1, dissolve sodium hyaluronate at a temperature of 40 - 45 °C. This temperature range can effectively avoid the degradation of hyaluronic acid while ensuring sufficient dissolution; a sodium hyaluronate concentration of 2 - 3% and a chlorhexidine concentration of 1 - 2% are the optimal reaction concentration ranges, which are conducive to improving the grafting efficiency; in step S3, a dripping rate of 2 - 3 mL / min combined with a stirring rate of 600 - 800 rpm can achieve sufficient contact of the reactants, while avoiding excessive local concentration and improving the reaction uniformity, and finally increasing the grafting rate of the product to more than 85%.

[0025] In step S1, the molecular weight of sodium hyaluronate is 1000 - 1500 kDa, the residual protein content of sodium hyaluronate is less than 0.1%, and the heavy metal residue content is less than 10 ppm. Specifically, the index setting of the molecular weight of sodium hyaluronate being 1000 - 1500 kDa and the purity not being less than 98% is based on the following mechanism: High molecular weight hyaluronic acid has more reaction sites, which is conducive to improving the grafting rate; while high purity can reduce impurity interference and avoid side reactions. Experiments have proved that when the molecular weight is lower than 1000 kDa, the water retention and viscosity of the product decrease significantly; while when it is higher than 1500 kDa, the viscosity of the reaction system is too large, which is not conducive to uniform reaction. The selection of this molecular weight range endows the product with ideal physical and chemical properties.

[0026] When preparing the sodium hyaluronate solution in step S1, one of the following stirring methods is adopted: Mechanical stirring at a rotation speed of 200 - 400 rpm; or Magnetic stirring at a rotation speed of 300 - 500 rpm; or Combined use of mechanical stirring and magnetic stirring, where the mechanical stirring rate is 200 - 400 rpm and the magnetic stirring rate is 300 - 500 rpm.

[0027] Specifically, the selective design of different stirring methods is based on the characteristics of the reaction system. Mechanical stirring (200 - 400 rpm) can provide a macroscopic mixing effect, while magnetic stirring (300 - 500 rpm) generates microscopic disturbances. When the two methods are used in combination, it can promote the contact of reactants at different scales. Experiments show that when using the combined stirring method, the grafting rate after 2 hours of reaction is 15 - 20% higher than that of a single stirring method, and the molecular weight distribution of the product is more concentrated (PDI is reduced to 1.3 - 1.5).

[0028] The ultrasonic dispersion conditions in step S2 are as follows: Ultrasonic power: 300 - 400 W; Ultrasonic frequency: 20 - 25 kHz; Working mode: intermittent ultrasound, and the ratio of working time to intermittent time is 3:1.

[0029] Specifically, the setting of ultrasonic dispersion parameters (power 300 - 400 W, frequency 20 - 25 kHz) is based on the study of the aggregation characteristics of chlorhexidine molecules. The ultrasonic energy under this condition is sufficient to break the aggregation between chlorhexidine molecules but will not cause molecular structure damage. The intermittent ultrasound mode (3:1 working ratio) can prevent the system temperature from being too high and ensure the dispersion effect. Experiments prove that the dispersion degree of the chlorhexidine solution under this condition is increased by more than 50%, laying a foundation for subsequent uniform grafting.

[0030] The specific conditions for the reaction in step S3 are as follows: Reaction temperature: 32 ± 0.5 °C; pH value: 6.8 - 7.2, adjusted by a buffer solution; Ionic strength of the reaction system: 0.1 - 0.15 mol / L; Reaction environment: nitrogen protection.

[0031] Specifically, the precise control of reaction conditions (temperature 32 ± 0.5 °C, pH 6.8 - 7.2, etc.) is based on reaction kinetics research: this temperature range can provide sufficient reaction activity while avoiding the degradation of hyaluronic acid; the pH value control is based on the ionization characteristics of chlorhexidine, and stable chemical bonding is facilitated within this range. Nitrogen protection can prevent oxidative degradation, and the control of ionic strength optimizes the reaction environment, increasing the product yield to over 85%.

[0032] In step S4: The amount of absolute ethanol used is 2 - 3 times the volume of the reaction mixture; The precipitation process is carried out in a low-temperature environment of 4 ± 1 °C; The precipitation process is carried out in the dark.

[0033] Specifically, the key parameters of the precipitation process (ethanol dosage 2 - 3 times, temperature 4 ± 1 °C, light avoidance) are because excessive ethanol can promote complete precipitation; and low temperature can slow down molecular movement and prevent agglomeration; light avoidance conditions prevent photosensitive reactions. The combination of these conditions improves the product purity to over 98%, and the particle size distribution is uniform (D90 ≤ 100 μm).

[0034] The washing process in step S5 includes: S51) Wash with 75% ethanol solution for the first time 2 times, 30 minutes each time; S52) Wash with 85% ethanol solution for the second time 2 times, 45 minutes each time; S53) Wash with 95% ethanol solution for the third time 1 time, for 60 minutes; The washing process is carried out under low-temperature and light-avoiding conditions.

[0035] Specifically, the design of the gradient washing process is based on the solubility characteristics of different impurities. 75% ethanol mainly removes water-soluble impurities, 85% removes medium-polarity substances, and 95% removes hydrophobic impurities. Successive washing with ethanol of different concentrations, combined with specific time, can achieve directional removal of impurities, reducing the free chlorhexidine content to below 0.5%.

[0036] The specific requirements for the drying process in step S6 are: S61) Heating stage: Raise the temperature from room temperature to 40 - 45 °C at a rate of 1 °C / min; S62) Constant-temperature stage: Maintain at 40 - 45 °C for 8 - 10 hours; S63) Cooling stage: Naturally cool to room temperature; S64) Vacuum degree: Gradually increase with the temperature and finally maintain at -0.08 MPa to -0.1 MPa.

[0037] Specifically, the staged drying process design is based on the thermodynamic characteristics of the product. Setting a heating rate of 1 °C / min can prevent heat shock; maintaining at 40 - 45 °C for 8 - 10 hours ensures sufficient drying; gradually increasing the vacuum degree avoids boiling, and finally controls the product moisture content to below 5% and there is no agglomeration phenomenon.

[0038] After step S6, the following determination steps are also included: S71) The molecular weight distribution and grafting rate of the product were determined by gel permeation chromatography; S72) The content of free chlorhexidine was determined by high performance liquid chromatography; S73) If the measurement results meet the following conditions, it is a qualified product: the molecular weight distribution is 800 - 1200 kDa, the grafting rate ≥ 85%, and the content of free chlorhexidine ≤ 0.5%; S74) If the above conditions are not met, the product is redissolved and purified through an ultrafiltration membrane with a cut-off molecular weight of 100 kDa. After collecting the retentate, steps S4 to S6 are repeated.

[0039] Specifically, establishing a multi-dimensional detection system can comprehensively characterize the product quality. The molecular weight distribution reflects the structural integrity, the grafting rate characterizes the modification effect, and the content of free monomers is related to the product safety. The ultrafiltration purification process for unqualified products can improve the product yield and reduce the production cost. This quality control system controls the batch-to-batch difference of the product within 3%.

[0040] According to another aspect of the present application, the following quality control indicators are also included: Q1) Residual amount of total volatile substances (LOD): ≤ 5.0% (w / w); Q2) Grafting rate determination (by gel permeation chromatography): ≥ 85.0%; Q3) Content of free monomers (by high performance liquid chromatography): ≤ 0.5% (w / w); Q4) Bacterial endotoxin (detected by limulus reagent method): ≤ 0.5 EU / mg.

[0041] According to still another aspect of the present application, the physicochemical property indicators of the chlorhexidine hyaluronate obtained by the preparation method are as follows: P1) Appearance: Off-white to slightly yellow amorphous powder; P2) Dissolution characteristics: Solubility ≥ 98% (w / v) in phosphate buffer solution with pH value of 6.5 - 7.5 for 24 h; P3) Particle size distribution (laser particle size analysis method): D90 ≤ 100 μm, and D50 is between 45 - 65 μm; P4) Accelerated stability: Stored for 30 days under the conditions of 60 ± 2 °C / 75 ± 5% RH, and the degradation rate of the effective substance content ≤ 5.0%.

[0042] Comparative Example 1: S1) Weigh 25 g of sodium hyaluronate (1200 kDa), dissolve it in 1000 mL of deionized water, and stir and dissolve it in a water bath at 42 °C for 2.5 hours to obtain a 2.5% sodium hyaluronate solution; S2) Dissolve 15 g of chlorhexidine digluconate in 1000 mL of deionized water, and stir and disperse it conventionally for 15 minutes to obtain a 1.5% chlorhexidine solution; S3) At 33 °C, with mechanical stirring (300 rpm), the chlorhexidine solution was added dropwise to the sodium hyaluronate solution at a rate of 5 mL / min, and the reaction was carried out for 3 hours; S4) 2000 mL of 95% ethanol was added to the reaction solution at once, and it was allowed to stand at room temperature for 4 hours; S5) Filtration was carried out, and it was washed 3 times with 95% ethanol, 500 mL each time; S6) Vacuum drying was carried out at 45 °C for 12 hours.

[0043] Table 1. Comprehensive evaluation of process parameters and product indicators of Comparative Example 1

[0044] Thus, from Comparative Example 1, it can be seen that: There are multiple key problems in the traditional process: 1) The temperature control accuracy is low, and the fluctuation range reaches ±3 °C, resulting in unstable reaction; 2) The single stirring method makes the reaction mixture insufficiently mixed, and there is a large local concentration difference; 3) The chlorhexidine is not sufficiently dispersed, and the average particle size is 5.2 μm, which is not conducive to uniform reaction; 4) The purification effect of ethanol with a single concentration is poor, and the residual amount of free chlorhexidine is as high as 1.8%; 5) Each product index does not meet the expected requirements. In particular, the grafting rate is only 72.3%, and the PDI is as high as 2.1, indicating a large structural inhomogeneity of the product. These problems directly lead to unstable product quality and large differences between batches, indicating that the existing technology urgently needs to be improved.

[0045] Comparative Example 2: S1) Weigh 25 g of 800 kDa sodium hyaluronate and dissolve it in 1000 mL of deionized water; Temperature control: 40 - 45 °C; Stirring method: Mechanical stirring at 700 rpm + magnetic stirring at 400 rpm; Stirring time: 2.5 hours; pH adjustment: 7.0 ± 0.2.

[0046] S2) Weigh 15 g of chlorhexidine digluconate and dissolve it in 1000 mL of deionized water; Ultrasonic conditions: 350 W, 22 kHz; Intermittent mode: Working for 3 min / stopping for 1 min; Total time: 18 min; Temperature control: 15 - 20 °C water bath.

[0047] S3) Reaction conditions are set as follows: Temperature: 32 ± 0.5 °C; pH: 7.0 ± 0.2; Stirring: 700 rpm mechanical + 400 rpm magnetic; Nitrogen protection; React for 3.5 hours.

[0048] S4) Post-treatment of the precipitate: Transfer the reaction solution to a low-temperature chamber at 4 ± 1 °C; Slowly add pre-cooled absolute ethanol dropwise at 2.5 times the volume; Dripping rate: 6 mL / min; Standing time: 5 hours; Under light-proof conditions.

[0049] S5) Purification process: Filter using a 0.45 μm pore size filter membrane; Wash with 75% ethanol twice, 40 min each time; Wash with 85% ethanol twice, 40 min each time; Wash with 95% ethanol once, 60 min; Operate under light-proof conditions at 4 °C throughout the process.

[0050] S6) Drying process: Heating rate: 1 °C / min; Drying temperature: 42 °C; Vacuum degree: gradually increase from -0.05 MPa to -0.09 Mpa; Drying time: 10 hours; Naturally cool to room temperature.

[0051] Table 2. Physicochemical indexes of the product of Comparative Example 2

[0052] Thus, from Comparative Example 2, it can be seen that: Using sodium hyaluronate with a low molecular weight (800 kDa) results in a significant decline in product performance, with the water retention capacity decreasing by 50% and the solution viscosity being only 65% of the standard product. This verifies the key influence of the raw material molecular weight on product performance and confirms the rationality of the molecular weight range of 1000 - 1500 kDa in the claims.

[0053] Example 1: S1) Weigh 25 g of sodium hyaluronate (1200 kDa, purity 99.2%) and dissolve it in 1000 mL of deionized water; Heat in a water bath to 42 °C and use a combination of mechanical stirring (350 rpm) and magnetic stirring (450 rpm) for 2.5 hours; Adjust the pH to 7.0 to obtain a 2.5% sodium hyaluronate solution.

[0054] S2) Weigh 15 g of chlorhexidine digluconate and dissolve it in 1000 mL of deionized water; Treat it by ultrasonic dispersion (350W, 22kHz, working for 3min / stopping for 1min) for 18min; Adjust the pH to 7.0 to obtain a 1.5% chlorhexidine solution.

[0055] S3) Control the temperature at 32.5°C and carry out the reaction under nitrogen protection: Dropwise add the chlorhexidine solution into the sodium hyaluronate solution at a rate of 2.5 mL / min; Combine mechanical stirring (700 rpm) with magnetic stirring (400 rpm); Monitor the pH in real time and maintain it at 7.0 ± 0.2; Adjust the ionic strength to 0.12 mol / L; React for 3.5 hours.

[0056] S4) Transfer the reaction solution to a low-temperature chamber at 4 ± 1°C: Slowly dropwise add 2.5 times the volume of anhydrous ethanol precooled to 4°C (dropwise addition rate 6 mL / min); Let it stand for 5 hours under light-shielded conditions.

[0057] S5) Filter and collect the precipitate, and carry out the following steps in sequence: Wash with 75% ethanol twice, 40 min each time; Wash with 85% ethanol twice, 40 min each time; Wash with 95% ethanol once, 60 min; Operate under light-shielded conditions at 4°C throughout the process.

[0058] S6) Control the drying process: Raise the temperature to 42°C at a rate of 1°C / min; Gradually increase the vacuum degree from -0.05 MPa to -0.09 MPa; Maintain for 10 hours; Naturally cool down to room temperature.

[0059] Table 3. Physical and chemical indexes of the product in Example 1

[0060] Thus, it can be seen from Example 1 that: The optimized process realizes the precise control of key parameters, and all indexes of the product meet the expected requirements: grafting rate 87.2%, PDI 1.42, free chlorhexidine 0.38%, and yield 83.5%. It proves the effectiveness of the process parameter optimization and provides a reliable basis for large-scale production.

[0061] Example 2: Continuously execute the process in Example 1 for 5 batches, with the same raw material dosage for each batch.

[0062] Table 4. Comparison of Physicochemical Indexes of Five Batches of Products in Example 2

[0063] Thus, it can be seen from Example 2 that: The relative standard deviations of 5 consecutive batches of production are all less than 3%, indicating that the process has excellent reproducibility. The key indexes such as grafting rate (86.8 - 87.5%) and PDI (1.42 - 1.46) have small fluctuations among batches, confirming the stability and controllability of the process.

[0064] Example 3: Take the product of Example 1 and store it under the conditions of 60°C / 75%RH, and conduct regular inspections (stability verification).

[0065] Table 5. Results of Accelerated Stability Test in Example 3

[0066] Thus, it can be seen from Example 3 that: The accelerated stability test shows that after 30 days of storage under the conditions of 60°C / 75%RH, the degradation rate of the product is only 4.2%, and all indexes remain stable. This indicates that the product has good thermal stability and storage stability, meeting the actual application requirements.

[0067] Example 4: Take the product obtained in Example 1 for biological evaluation.

[0068] Antibacterial activity test: The minimum inhibitory concentration (MIC) was determined by the microbroth dilution method, using unmodified chlorhexidine as a control.

[0069] Test strains: Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922).

[0070] Cultivation conditions: 37°C, 24 hours.

[0071] Cytotoxicity test: The cytotoxicity to human fibroblasts (L929) was evaluated by the CCK-8 method.

[0072] Cell culture conditions: 37°C, 5% CO2, culture for 48 hours.

[0073] Set concentration gradients: 0, 50, 100, 150, 200, 250 μg / mL.

[0074] Table 6. Results of Biological Evaluation in Example 4

[0075] Thus, it can be seen from Example 4 that: The biological evaluation results show that the product has excellent antibacterial activity (the MIC value is reduced by 50%) and good biocompatibility (IC50 > 200 μg / mL). Safety indicators such as cytotoxicity and hemolysis are better than those of the control product, confirming the safety and effectiveness of the product.

[0076] Example 5: Chlorhexidine digluconate was processed under the ultrasonic conditions of Example 1 to investigate the dispersion effect.

[0077] Dispersion process parameters: Ultrasonic power: 350 W Frequency: 22 kHz Intermittent mode: Working for 3 min / stopping for 1 min Total time: 18 min Temperature control: 15 - 20°C water bath Table 7. Evaluation of the dispersion effect in Example 5

[0078] Thus, it can be seen from Example 5 that: The ultrasonic dispersion process reduces the average particle size of chlorhexidine from 5.2 μm to 2.6 μm, and significantly improves the dispersion stability. The Zeta potential increases by 82.1%, confirming the stability of the dispersion system and laying a foundation for improving the product quality.

[0079] It should be noted that all the data in the above examples are the averages of 3 repeated experiments, and the relative standard deviations are all less than 5%. The experimental results show that the process of the present invention has good repeatability and stability, and the product performance is significantly better than the prior art.

[0080] Specifically, through systematic process optimization and strict quality control, the present invention has successfully solved various problems existing in the prior art. All indicators of the product are better than those of the control, with significant innovation and practical value, and are suitable for industrial production.

[0081] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of chlorhexidine hyaluronate with bacteriostatic effect, characterized in that, It includes the following steps: S1) Dissolve sodium hyaluronate in deionized water, stir and dissolve at 40 - 45 °C for 2 - 3 hours to prepare a sodium hyaluronate solution with a mass fraction of 2 - 3%, and then cool it to room temperature; S2) Dissolve chlorhexidine digluconate in deionized water, and ultrasonically disperse it for 15 - 20 minutes to prepare a chlorhexidine digluconate solution with a mass fraction of 1 - 2%; S3) At 30 - 35 °C, slowly drop the chlorhexidine digluconate solution in step S2 into the sodium hyaluronate solution in step S1 at a dropping rate of 2 - 3 mL / min, while maintaining a stirring rate of 600 - 800 rpm. After the dropping is completed, continue to stir and react for 2 - 4 hours; S4) Slowly add pre-cooled absolute ethanol at 4 °C to the reaction mixture at a rate of 5 - 8 mL / min, and let it stand for precipitation for 4 - 6 hours; S5) Filter and collect the precipitate with a filter membrane with a pore size of 0.45 μm, and repeatedly wash it 3 - 5 times with ethanol solutions with a volume ratio of 75%, 85%, and 95% in sequence, and each washing time is not less than 30 minutes; S6) Place the obtained precipitate in a vacuum drying oven at 40 - 45 °C, heat it up at a heating rate of 1 °C / min, dry it for 8 - 12 hours, and maintain the vacuum degree at -0.08 MPa to -0.1 MPa to obtain chlorhexidine hyaluronate.

2. The preparation method of chlorhexidine hyaluronate according to claim 1, characterized in that, In step S1, the molecular weight of sodium hyaluronate is 1000 - 1500 kDa, the residual protein content of sodium hyaluronate is less than 0.1%, and the heavy metal residue content is less than 10 ppm.

3. The preparation method of chlorhexidine hyaluronate according to claim 1, characterized in that, When preparing the sodium hyaluronate solution in step S1, one of the following stirring methods is adopted: Mechanical stirring, with a rotation speed of 200 - 400 rpm; or Magnetic stirring, with a rotation speed of 300 - 500 rpm; or Combined use of mechanical stirring and magnetic stirring, where the mechanical stirring rate is 200 - 400 rpm and the magnetic stirring rate is 300 - 500 rpm.

4. The preparation method of chlorhexidine hyaluronate according to claim 1, wherein The ultrasonic dispersion conditions in step S2 are: Ultrasonic power: 300 - 400 W; Ultrasonic frequency: 20 - 25 kHz; Working mode: intermittent ultrasound, and the ratio of working time to intermittent time is 3:

1.

5. The preparation method of chlorhexidine hyaluronate according to claim 1, characterized in that, The specific conditions of the reaction in step S3 are: Reaction temperature: 32 ± 0.5 °C; pH value: 6.8 - 7.2, adjusted by a buffer solution; Ionic strength of the reaction system: 0.1 - 0.15 mol / L; Reaction environment: nitrogen protection.

6. The preparation method of chlorhexidine hyaluronate according to claim 1, characterized in that, In step S4: The dosage of absolute ethanol is 2 - 3 times the volume of the reaction mixture; The precipitation process is carried out in a low-temperature environment at 4 ± 1 °C; The precipitation process is carried out in the dark.

7. The preparation method of chlorhexidine hyaluronate according to claim 1, characterized in that, The washing process in step S5 includes: S51) Wash twice with 75% ethanol solution for 30 minutes each time; S52) Wash twice with 85% ethanol solution for 45 minutes each time; S53) Wash once with 95% ethanol solution for 60 minutes; The washing process is carried out under low-temperature and light-proof conditions.

8. The preparation method of chlorhexidine hyaluronate according to claim 1, wherein The specific requirements for the drying process in step S6 are: S61) Heating stage: Heat up from room temperature to 40 - 45 °C at a rate of 1 °C / min; S62) Constant temperature stage: Maintain at 40 - 45 °C for 8 - 10 hours; S63) Cooling stage: Naturally cool to room temperature; S64) Vacuum degree: Gradually increase with temperature and finally maintain at -0.08 MPa to -0.1 MPa.

9. The preparation method of chlorhexidine hyaluronate according to claim 1, characterized in that, After the step S6, the following determination steps are further included: S71) Use gel permeation chromatography to determine the molecular weight distribution and grafting rate of the product; S72) Use high performance liquid chromatography to determine the content of free chlorhexidine; S73) If the determination results meet the following conditions, it is a qualified product: the molecular weight distribution is 800 - 1200 kDa, the grafting rate ≥ 85%, and the content of free chlorhexidine ≤ 0.5%; S74) If the above conditions are not met, redissolve the product, purify it through a ultrafiltration membrane with a molecular weight cut-off of 100 kDa, collect the retentate, and repeat steps S4 to S6.

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