Preparation method of hyaluronic acid chlorhexidine with bacteriostatic effect

Abstract

The application discloses a preparation method of hyaluronic acid chlorhexidine with bacteriostatic effect, and comprises the following steps: S1) preparing a sodium hyaluronate solution with a mass fraction of 2-3%; S2) preparing a chlorhexidine digluconate solution with a mass fraction of 1-2%; S3) slowly adding the chlorhexidine digluconate solution in the step S2) into the sodium hyaluronate solution in the step S1) and stirring to react; S4) adding anhydrous ethanol pre-cooled to 4 DEG C into the reaction mixture and standing to precipitate; S5) filtering the precipitate by using a filter membrane and repeatedly washing the precipitate with ethanol solutions with volume ratios of 75%, 85% and 95% for 3-5 times in sequence; and S6) drying the obtained precipitate for 8-12 hours to obtain hyaluronic acid chlorhexidine. The application adopts specific process parameters and reaction conditions, controls the reaction temperature, pH value and ionic strength accurately, and combines the synergistic effect of mechanical stirring and magnetic stirring, so that the uniformity and controllability of the reaction are remarkably improved, and the grafting rate of the hyaluronic acid chlorhexidine product prepared by the method can reach more than 85%.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for preparing chlorhexidine hyaluronic acid with antibacterial properties. Background Technology

[0002] Chlorhexidine hyaluronic acid is a novel antibacterial material prepared by chemically modifying hyaluronic acid with chlorhexidine. It combines the biocompatibility of hyaluronic acid with the broad-spectrum antibacterial properties of chlorhexidine, showing great promise for applications in medical devices, tissue engineering, and drug delivery. Currently, it is mainly prepared by reacting sodium hyaluronate with chlorhexidine digluconate, a method that offers advantages such as readily available raw materials and mild reaction conditions. Existing technologies commonly use preparation methods including chemical cross-linking, physical adsorption, and electrostatic bonding, with chemical cross-linking being widely used due to its good binding stability and excellent product performance. Furthermore, studies have shown that chlorhexidine hyaluronic acid exhibits more durable antibacterial activity and lower cytotoxicity compared to chlorhexidine alone.

[0003] However, the following key problems exist in the existing technology:

[0004] (1) Temperature and pH control issues: Traditional processes use simple heating devices and intermittent pH monitoring, which cannot achieve precise temperature control and real-time pH monitoring. When the temperature of the reaction system fluctuates by more than ±2℃, it will cause the hyaluronic acid chain segments to break; and when the pH value deviates from the neutral range (pH 6.8-7.2), it will affect the grafting efficiency of chlorhexidine. These factors ultimately lead to an excessively wide molecular weight distribution range of the product (PDI>2.0), affecting product performance.

[0005] (2) Defects in purification process: Currently, the common method in industrial production is to use a single precipitation with 95% ethanol for purification. Although this method is simple to operate, the low solubility of free chlorhexidine in high-concentration ethanol makes it difficult to remove completely. At the same time, hyaluronic acid tends to aggregate in high-concentration ethanol, resulting in poor product purification and a residual amount of free chlorhexidine that is usually above 2%.

[0006] (3) Insufficient control of the drying process: Existing drying processes mostly use constant temperature drying (e.g., 45℃) and lack temperature gradient control. Due to the heat sensitivity of chlorhexidine hyaluronic acid, if the temperature rises too quickly in the early stage of drying, it will lead to local overheating and degradation of the product; and if the vacuum degree is not properly controlled in the later stage, it will cause residual moisture inside the product and cause agglomeration. These problems seriously affect the solubility and stability of the product.

[0007] (4) Inadequate quality evaluation system: Existing technologies mainly rely 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 leads to the inability to detect and resolve product quality problems in a timely manner, resulting in large batch-to-batch differences and making it difficult to guarantee product quality. According to statistics, the relative standard deviation of the grafting rate between batches of products produced using traditional methods can reach more than 15%.

[0008] Therefore, there is an urgent need to develop a method for preparing chlorhexidine hyaluronic acid that can precisely control reaction conditions, optimize purification processes, and improve product quality stability. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing chlorhexidine hyaluronic acid, so as to solve the problems existing in the current methods for preparing chlorhexidine hyaluronic acid as mentioned in the background art.

[0010] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing chlorhexidine hyaluronic acid with antibacterial activity, comprising the following steps:

[0011] S1) Dissolve sodium hyaluronate in deionized water and stir at 40-45℃ for 2-3 hours to prepare a sodium hyaluronate solution with a mass fraction of 2-3%, and then cool it to room temperature;

[0012] S2) Dissolve chlorhexidine digluconate in deionized water and ultrasonically disperse for 15-20 minutes to prepare a chlorhexidine digluconate solution with a mass fraction of 1-2%.

[0013] S3) At 30-35℃, slowly add the chlorhexidine digluconate solution from step S2 to the sodium hyaluronate solution from step S1 at a dropping rate of 2-3 mL / min, while maintaining a stirring rate of 600-800 rpm. After the addition is complete, continue stirring and reacting for 2-4 hours.

[0014] S4) Slowly add anhydrous ethanol pre-cooled to 4°C to the reaction mixture at a rate of 5-8 mL / min, and let it stand for 4-6 hours to precipitate.

[0015] S5) Collect the precipitate by filtration with a filter membrane with a pore size of 0.45 μm, and wash it repeatedly with ethanol solutions of 75%, 85%, and 95% by volume 3-5 times, with each washing time not less than 30 minutes.

[0016] S6) The obtained precipitate was placed in a vacuum drying oven at 40-45℃ and heated at a rate of 1℃ / min for 8-12 hours, while maintaining the vacuum at -0.08MPa to -0.1MPa to obtain chlorhexidine hyaluronic acid.

[0017] 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 residual heavy metal content is less than 10 ppm.

[0018] Preferably, in step S1, when preparing the sodium hyaluronate solution, one of the following stirring methods is used:

[0019] Mechanical stirring, at a speed of 200-400 rpm; or

[0020] Magnetic stirring, at a speed of 300-500 rpm; or

[0021] Mechanical stirring and magnetic stirring are used in combination, with the mechanical stirring speed being 200-400 rpm and the magnetic stirring speed being 300-500 rpm.

[0022] Preferably, the ultrasonic dispersion conditions in step S2 are as follows:

[0023] Ultrasonic power: 300-400W;

[0024] Ultrasonic frequency: 20-25kHz;

[0025] Working mode: intermittent ultrasound, with a working time to intermittent time ratio of 3:1.

[0026] Preferably, the specific conditions for the reaction in step S3 are as follows:

[0027] Reaction temperature: 32±0.5℃;

[0028] pH value: 6.8-7.2, adjusted by buffer solution;

[0029] The ionic strength of the reaction system is 0.1-0.15 mol / L.

[0030] Reaction environment: Nitrogen protection.

[0031] Preferably, in step S4:

[0032] The amount of anhydrous ethanol used is 2-3 times the volume of the reaction mixture;

[0033] The precipitation process was carried out at a low temperature of 4±1℃;

[0034] The sedimentation process should be kept away from light.

[0035] Preferably, the washing process in step S5 includes:

[0036] S51) Wash twice with 75% ethanol solution for the first time, 30 minutes each time;

[0037] S52) Wash twice with 85% ethanol solution, 45 minutes each time;

[0038] S53) Wash once with 95% ethanol solution for 60 minutes;

[0039] The washing process is carried out under low temperature and light-protected conditions.

[0040] Preferably, the specific requirements for the drying process in step S6 are as follows:

[0041] S61) Heating stage: Increase the temperature from room temperature to 40-45℃ at a rate of 1℃ / min;

[0042] S62) Constant temperature stage: Maintain at 40-45℃ for 8-10 hours;

[0043] S63) Cooling stage: Natural cooling to room temperature;

[0044] S64) Vacuum degree: As the temperature gradually increases, it eventually remains at -0.08MPa to -0.1MPa.

[0045] Preferably, the following measurement step is included after step S6:

[0046] S71) The molecular weight distribution and grafting rate of the product were determined by gel permeation chromatography;

[0047] S72) The content of free chlorhexidine was determined by high performance liquid chromatography;

[0048] S73) If the test results meet the following conditions, the product is considered qualified: molecular weight distribution is 800-1200 kDa, grafting rate is ≥85%, and free chlorhexidine content is ≤0.5%;

[0049] S74) If the above conditions are not met, the product is redissolved and purified by ultrafiltration with a molecular weight cutoff of 100 kDa. After collecting the retentate, steps S4 to S6 are repeated.

[0050] Compared with the prior art, the beneficial effects of the present invention are:

[0051] 1) This application employs specific process parameters and reaction conditions. By precisely controlling the reaction temperature, pH value, and ionic strength, and combining the synergistic effect of mechanical and magnetic stirring, the uniformity and controllability of the reaction are significantly improved. Experimental results show that the grafting rate of chlorhexidine hyaluronic acid product prepared by this method can reach over 85%, with a uniform molecular weight distribution (polydispersity index PDI ≤ 1.5) and a free chlorhexidine content of less than 0.5%. Compared with the prior art, this invention significantly improves the uniformity and purity of the product, laying the foundation for subsequent applications. By regulating the ionic strength of the reaction system and using nitrogen protection, product degradation is effectively prevented, and the reaction yield is improved. Experimental data show that under the same raw material dosage conditions, the product yield of this invention is 15-20% higher than that of existing methods.

[0052] 2) This application employs an innovative gradient ethanol solution purification and precise temperature-controlled drying process, effectively solving key issues in product purification and drying. Through stepwise purification with 75%, 85%, and 95% ethanol, combined with low-temperature and light-protected conditions, not only is the product purity significantly improved, but the structural integrity of the product is also maintained to the greatest extent. Compared with traditional single-concentration ethanol purification, the gradient purification method of this invention can increase product purity by 10-15%, while effectively reducing the amount of organic solvent used. The combination of programmed heating and vacuum drying achieves gentle drying of the product, effectively preventing agglomeration and degradation during the drying process. Thermogravimetric analysis results show that the product prepared by this invention has excellent thermal stability; after storage at 60℃ for 30 days, the content degradation rate does not exceed 5%.

[0053] 3) This application establishes a comprehensive product quality control system, employing advanced analytical methods such as gel permeation chromatography and high-performance liquid chromatography to fully characterize the product, and sets clear quality control indicators. When product quality fails to meet standards, it can be reprocessed through ultrafiltration purification to ensure stable and controllable final product quality. This quality control system not only guarantees product quality consistency but also significantly improves production efficiency. Experiments have demonstrated that this method has good batch-to-batch reproducibility, with a relative standard deviation (RSD) of less than 3%, stable product performance, and fully meets pharmaceutical-grade quality requirements. The process route of this invention is simple, easy to operate, requires minimal equipment investment, and has low raw material costs, making it suitable for industrial production. Using the method of this invention, the large-scale production of chlorhexidine hyaluronic acid can be achieved, providing reliable process assurance for its application in medical devices, biomaterials, and other fields.

[0054] 4) The chlorhexidine hyaluronic acid prepared in this application also exhibits excellent biological properties. In vitro antibacterial experiments show that the minimum inhibitory concentration (MIC) of this product against Staphylococcus aureus and Escherichia coli is 4 μg / mL and 8 μg / mL, respectively, which is significantly lower than that of unmodified chlorhexidine. The cytotoxicity test results show that the half-maximal inhibitory concentration (IC50) of the product against human fibroblasts is higher than 200 μg / mL, indicating good biocompatibility. These excellent biological properties lay the foundation for the application of the product in wound dressings, anti-infection materials and other fields. Attached Figure Description

[0055] Figure 1 This is a flowchart of the preparation method of this application. Detailed Implementation

[0056] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0057] In the description of the invention, it should be noted that the execution order of the steps is not limited by the sequence number. The possible changes in the order of some steps, the synchronous execution of steps, and the split execution of steps are all within the scope of protection of this application.

[0058] Please see Figure 1 This invention provides a technical solution: a method for preparing chlorhexidine hyaluronic acid, comprising the following steps:

[0059] S1) Dissolve sodium hyaluronate in deionized water and stir at 40-45℃ for 2-3 hours to prepare a sodium hyaluronate solution with a mass fraction of 2-3%, and then cool it to room temperature;

[0060] S2) Dissolve chlorhexidine digluconate in deionized water and ultrasonically disperse for 15-20 minutes to prepare a chlorhexidine digluconate solution with a mass fraction of 1-2%.

[0061] S3) At 30-35℃, slowly add the chlorhexidine digluconate solution from step S2 to the sodium hyaluronate solution from step S1 at a dropping rate of 2-3 mL / min, while maintaining a stirring rate of 600-800 rpm. After the addition is complete, continue stirring and reacting for 2-4 hours.

[0062] S4) Slowly add anhydrous ethanol pre-cooled to 4°C to the reaction mixture at a rate of 5-8 mL / min, and let it stand for 4-6 hours to precipitate.

[0063] S5) Collect the precipitate by filtration with a filter membrane with a pore size of 0.45 μm, and wash it repeatedly with ethanol solutions of 75%, 85%, and 95% by volume 3-5 times, with each washing time not less than 30 minutes.

[0064] S6) The obtained precipitate was placed in a vacuum drying oven at 40-45℃ and heated at a rate of 1℃ / min for 8-12 hours, while maintaining the vacuum at -0.08MPa to -0.1MPa to obtain chlorhexidine hyaluronic acid.

[0065] Specifically, by precisely controlling the process parameters of each preparation step from S1 to S6, a comprehensive improvement in product quality was achieved.

[0066] Specifically, in step S1, sodium hyaluronate is dissolved at a temperature of 40-45℃. This temperature range effectively prevents hyaluronic acid degradation while ensuring complete dissolution. A concentration of 2-3% sodium hyaluronate and 1-2% chlorhexidine is the optimal reaction concentration range, which is beneficial for improving grafting efficiency. In step S3, a dropping rate of 2-3 mL / min combined with a stirring rate of 600-800 rpm ensures sufficient contact of the reactants while avoiding excessively high local concentrations, thus improving reaction uniformity and ultimately increasing the grafting rate of the product to over 85%.

[0067] In step S1, the molecular weight of sodium hyaluronate is 1000-1500 kDa, the residual protein content is less than 0.1%, and the heavy metal residue is less than 10 ppm. Specifically, the molecular weight of sodium hyaluronate in the 1000-1500 kDa range and the purity of not less than 98% are set based on the following mechanism: high molecular weight hyaluronic acid has more reaction sites, which is beneficial to improving the grafting rate; while high purity can reduce impurity interference and avoid side reactions. Experiments have shown that when the molecular weight is below 1000 kDa, the water-holding capacity and viscosity of the product decrease significantly; while when it is above 1500 kDa, the viscosity of the reaction system is too high, which is not conducive to uniform reaction. The selection of this molecular weight range gives the product ideal physicochemical properties.

[0068] When preparing the sodium hyaluronate solution in step S1, one of the following stirring methods shall be used:

[0069] Mechanical stirring, at a speed of 200-400 rpm; or

[0070] Magnetic stirring, at a speed of 300-500 rpm; or

[0071] Mechanical stirring and magnetic stirring are used in combination, with the mechanical stirring speed being 200-400 rpm and the magnetic stirring speed being 300-500 rpm.

[0072] Specifically, the selective design of different stirring methods is based on the characteristics of the reaction system. Mechanical stirring (200-400 rpm) provides macroscopic mixing effects, while magnetic stirring (300-500 rpm) generates microscopic disturbances. When the two methods are used in combination, they can promote contact of reactants at different scales. Experiments show that using the combined stirring method, the grafting rate after 2 hours of reaction is 15-20% higher than that of the single stirring method, and the molecular weight distribution of the product is more concentrated (PDI is reduced to 1.3-1.5).

[0073] The ultrasonic dispersion conditions in step S2 are as follows:

[0074] Ultrasonic power: 300-400W;

[0075] Ultrasonic frequency: 20-25kHz;

[0076] Working mode: intermittent ultrasound, with a working time to intermittent time ratio of 3:1.

[0077] Specifically, the ultrasonic dispersion parameters (power 300-400W, frequency 20-25kHz) were set based on studies of the aggregation characteristics of chlorhexidine molecules. Under these conditions, the ultrasonic energy is sufficient to break up the aggregation of chlorhexidine molecules without causing molecular structure damage. Intermittent ultrasonication (3:1 working ratio) prevents excessively high system temperature, ensuring effective dispersion. Experiments have shown that under these conditions, the dispersion of the chlorhexidine solution is improved by more than 50%, laying the foundation for subsequent uniform grafting.

[0078] The specific conditions for the reaction in step S3 are as follows:

[0079] Reaction temperature: 32±0.5℃;

[0080] pH value: 6.8-7.2, adjusted by buffer solution;

[0081] The ionic strength of the reaction system is 0.1-0.15 mol / L.

[0082] Reaction environment: Nitrogen protection.

[0083] Specifically, the precise control of reaction conditions (temperature 32±0.5℃, pH 6.8-7.2, etc.) is based on reaction kinetic studies: this temperature range provides sufficient reactivity while avoiding hyaluronic acid degradation; pH control is based on the ionization characteristics of chlorhexidine, which favors the formation of stable chemical bonds within this range. Nitrogen protection prevents oxidative degradation, and ionic strength control optimizes the reaction environment, increasing product yield to over 85%.

[0084] In step S4:

[0085] The amount of anhydrous ethanol used is 2-3 times the volume of the reaction mixture;

[0086] The precipitation process was carried out at a low temperature of 4±1℃;

[0087] The sedimentation process should be kept away from light.

[0088] Specifically, the key parameters for the precipitation process (2-3 times the amount of ethanol, temperature 4±1℃, and protection from light) are as follows: excess ethanol promotes complete precipitation; low temperature slows down molecular motion and prevents aggregation; and the protection from light prevents photosensitive reactions. The combination of these conditions increases the product purity to over 98% and ensures uniform particle distribution (D90≤100μm).

[0089] The washing process in step S5 includes:

[0090] S51) Wash twice with 75% ethanol solution for the first time, 30 minutes each time;

[0091] S52) Wash twice with 85% ethanol solution, 45 minutes each time;

[0092] S53) Wash once with 95% ethanol solution for 60 minutes;

[0093] The washing process is carried out under low temperature and light-protected conditions.

[0094] Specifically, the gradient washing process is designed based on the solubility characteristics of different impurities. 75% ethanol mainly removes water-soluble impurities, 85% removes moderately polar substances, and 95% removes hydrophobic impurities. Washing with different concentrations of ethanol in stages, combined with specific time intervals, achieves targeted removal of impurities, reducing the free chlorhexidine content to below 0.5%.

[0095] The specific requirements for the drying process in step S6 are as follows:

[0096] S61) Heating stage: Increase the temperature from room temperature to 40-45℃ at a rate of 1℃ / min;

[0097] S62) Constant temperature stage: Maintain at 40-45℃ for 8-10 hours;

[0098] S63) Cooling stage: Natural cooling to room temperature;

[0099] S64) Vacuum degree: As the temperature gradually increases, it eventually remains at -0.08MPa to -0.1MPa.

[0100] Specifically, the staged drying process is designed based on the thermodynamic characteristics of the product. Setting a heating rate of 1℃ / min can prevent heat shock; maintaining 40-45℃ for 8-10 hours ensures thorough drying; the vacuum degree is gradually increased to avoid boiling, ultimately controlling the product moisture content to below 5% and preventing agglomeration.

[0101] Step S6 is followed by the following measurement steps:

[0102] S71) The molecular weight distribution and grafting rate of the product were determined by gel permeation chromatography;

[0103] S72) The content of free chlorhexidine was determined by high performance liquid chromatography;

[0104] S73) If the test results meet the following conditions, the product is considered qualified: molecular weight distribution is 800-1200 kDa, grafting rate is ≥85%, and free chlorhexidine content is ≤0.5%;

[0105] S74) If the above conditions are not met, the product is redissolved and purified by ultrafiltration with a molecular weight cutoff of 100 kDa. After collecting the retentate, steps S4 to S6 are repeated.

[0106] Specifically, establishing a multi-dimensional testing system can comprehensively characterize product quality. Molecular weight distribution reflects structural integrity, grafting rate characterizes modification effect, and free monomer content relates to product safety. Ultrafiltration purification of substandard products can improve product yield and reduce production costs. This quality control system keeps batch-to-batch variations within 3%.

[0107] According to another aspect of this application, the following quality control indicators are also included:

[0108] Q1) Total Volatile Organic Compounds Residue (LOD): ≤5.0% (w / w);

[0109] Q2) Grafting rate determination (by gel permeation chromatography): ≥85.0%;

[0110] Q3) Free monomer content (by high performance liquid chromatography): ≤0.5% (w / w);

[0111] Q4) Bacterial endotoxins (detected by the Limulus amebocyte lysate (LAL) reagent method): ≤0.5 EU / mg.

[0112] According to another aspect of this application, the physicochemical properties of chlorhexidine hyaluronic acid obtained by the preparation method are as follows:

[0113] P1) Appearance: Off-white to slightly yellow amorphous powder;

[0114] P2) Solubility characteristics: Solubility in phosphate buffer solution with pH 6.5-7.5 ≥98% (w / v) after 24h;

[0115] P3) Particle size distribution (laser particle size analysis): D90≤100μm, and D50 is between 45-65μm;

[0116] P4) Accelerated stability: After storage at 60±2℃ / 75±5%RH for 30 days, the degradation rate of effective substances is ≤5.0%.

[0117] Comparative Example 1: S1) 25g of sodium hyaluronate (1200kDa) was weighed and dissolved in 1000mL of deionized water. The solution was stirred in a water bath at 42℃ for 2.5 hours to obtain a 2.5% sodium hyaluronate solution.

[0118] S2) Dissolve 15g of chlorhexidine digluconate in 1000mL of deionized water and disperse by stirring for 15 minutes to prepare a 1.5% chlorhexidine solution;

[0119] S3) At 33℃, chlorhexidine solution was added dropwise to sodium hyaluronate solution at a rate of 5 mL / min using mechanical stirring (300 rpm) and the reaction was carried out for 3 hours.

[0120] S4) Add 2000 mL of 95% ethanol to the reaction solution at once and let it stand at room temperature for 4 hours;

[0121] S5) Filter by suction, wash three times with 95% ethanol, 500 mL each time;

[0122] S6) Vacuum dry at 45℃ for 12 hours.

[0123] Table 1. Comprehensive Evaluation of Process Parameters and Product Indicators in Comparative Example 1

[0124]

[0125] Therefore, by comparing with Example 1, we can see that:

[0126] Traditional processes suffer from several key problems: 1) Low temperature control precision, with fluctuations ranging from ±3℃, leading to reaction instability; 2) Insufficient mixing due to a single stirring method, resulting in large local concentration differences; 3) Inadequate dispersion of chlorhexidine, with an average particle size of 5.2μm, hindering uniform reaction; 4) Poor purification effect with a single concentration of ethanol, resulting in a free chlorhexidine residue as high as 1.8%; 5) Failure to meet expected product indicators, particularly a grafting rate of only 72.3% and a PDI as high as 2.1, indicating significant product structural inhomogeneity. These problems directly lead to unstable product quality and large batch-to-batch variations, highlighting the urgent need for improvement in existing technologies.

[0127] Comparative Example 2: S1) Weigh 25g of 800kDa sodium hyaluronate and dissolve it in 1000mL of deionized water;

[0128] Temperature control: 40-45℃;

[0129] Mixing method: Mechanical stirring at 700 rpm + magnetic stirring at 400 rpm;

[0130] Mixing time: 2.5 hours;

[0131] pH adjustment: 7.0±0.2.

[0132] S2) Weigh 15g of chlorhexidine digluconate and dissolve it in 1000mL of deionized water;

[0133] Ultrasonic conditions: 350W, 22kHz;

[0134] Intermittent mode: Work for 3 minutes / Stop for 1 minute;

[0135] Total time: 18 minutes;

[0136] Temperature control: 15-20℃ water bath.

[0137] S3) Reaction conditions setting:

[0138] Temperature: 32±0.5℃;

[0139] pH: 7.0±0.2;

[0140] Stirring: 700 rpm mechanical + 400 rpm magnetic;

[0141] Nitrogen protection;

[0142] The reaction lasted 3.5 hours.

[0143] S4) Post-treatment precipitation:

[0144] Transfer the reaction solution to a low-temperature chamber at 4±1℃;

[0145] Slowly add pre-cooled anhydrous ethanol dropwise at a volume of 2.5.

[0146] Dropping rate: 6 mL / min;

[0147] Setting time: 5 hours;

[0148] Light-protected conditions.

[0149] S5) Purification process:

[0150] Filtration is performed using a 0.45μm pore size filter membrane;

[0151] Wash twice with 75% ethanol, 40 minutes each time;

[0152] Wash twice with 85% ethanol, 40 minutes each time;

[0153] Wash once with 95% ethanol for 60 minutes;

[0154] Operate at 4℃ and away from light throughout the entire process.

[0155] S6) Drying process:

[0156] Heating rate: 1℃ / min;

[0157] Drying temperature: 42℃;

[0158] Vacuum degree: gradually increased from -0.05MPa to -0.09MPa;

[0159] Drying time: 10 hours;

[0160] Let it cool naturally to room temperature.

[0161] Table 2. Physicochemical properties of Comparative Example 2 products

[0162]

[0163] Therefore, by comparing Example 2, we can see that:

[0164] Using low molecular weight (800kDa) sodium hyaluronate resulted in a significant decrease in product performance, with water retention reduced by 50% and solution viscosity only 65% ​​of the standard. This verified the critical impact of raw material molecular weight on product performance and confirmed the rationality of the 1000-1500kDa molecular weight range in the claims.

[0165] Example 1: S1) Weigh 25g of sodium hyaluronate (1200kDa, purity 99.2%) and dissolve it in 1000mL of deionized water;

[0166] Heat the water bath to 42°C and use a combination of mechanical stirring (350 rpm) and magnetic stirring (450 rpm) for 2.5 hours.

[0167] Adjust the pH to 7.0 to obtain a 2.5% sodium hyaluronate solution.

[0168] S2) Weigh 15g of chlorhexidine digluconate and dissolve it in 1000mL of deionized water;

[0169] The sample was treated with ultrasonic dispersion (350W, 22kHz, 3 min on, 1 min off) for 18 min.

[0170] Adjust the pH to 7.0 to prepare a 1.5% chlorhexidine solution.

[0171] S3) The reaction is carried out at a temperature controlled at 32.5℃ under nitrogen protection:

[0172] Chlorhexidine solution was added dropwise to sodium hyaluronate solution at a rate of 2.5 mL / min;

[0173] Mechanical stirring (700 rpm) and magnetic stirring (400 rpm) are used in combination;

[0174] pH was monitored in real time and maintained at 7.0±0.2;

[0175] The ionic strength was adjusted to 0.12 mol / L;

[0176] The reaction lasted 3.5 hours.

[0177] S4) Transfer the reaction solution to a low-temperature chamber at 4±1℃:

[0178] Slowly add 2.5 times the volume of anhydrous ethanol pre-cooled to 4°C (dropping rate 6 mL / min).

[0179] Let it stand for 5 hours in a dark place.

[0180] S5) Collect the precipitate by vacuum filtration, and proceed as follows:

[0181] Wash twice with 75% ethanol, 40 minutes each time;

[0182] Wash twice with 85% ethanol, 40 minutes each time;

[0183] Wash once with 95% ethanol for 60 minutes;

[0184] Operate at 4℃ and away from light throughout the entire process.

[0185] S6) Drying process control:

[0186] Increase the temperature by 1℃ / min to 42℃;

[0187] The vacuum level was gradually increased from -0.05 MPa to -0.09 MPa;

[0188] Maintain for 10 hours;

[0189] Let it cool naturally to room temperature.

[0190] Table 3. Physicochemical properties of the product from Example 1

[0191]

[0192] Therefore, as can be seen from Example 1:

[0193] The optimized process achieved precise control of key parameters, and all product indicators met the expected requirements: grafting rate 87.2%, PDI 1.42, free chlorhexidine 0.38%, and yield 83.5%. This demonstrates the effectiveness of the optimized process parameters and provides a reliable basis for large-scale production.

[0194] Example 2: Five consecutive batches were processed according to the process in Example 1, with the same amount of raw materials used in each batch.

[0195] Table 4. Comparison of Physicochemical Indicators of Five Batches of Products in Example 2

[0196]

[0197] Therefore, as can be seen from Example 2:

[0198] The relative standard deviation of five consecutive production batches was less than 3%, indicating that the process has excellent reproducibility. Key indicators such as grafting rate (86.8-87.5%) and PDI (1.42-1.46) showed small fluctuations between batches, confirming the stability and controllability of the process.

[0199] Example 3: Take the product from Example 1 and store it at 60℃ / 75%RH, and test it periodically (stability verification).

[0200] Table 5. Accelerated stability test results of Example 3

[0201]

[0202] Therefore, as can be seen from Example 3:

[0203] Accelerated stability testing showed that after 30 days of storage at 60℃ / 75%RH, the product degradation rate was only 4.2%, and all indicators remained stable. This indicates that the product has good thermal and storage stability, meeting practical application requirements.

[0204] Example 4: Biological evaluation was performed on the product obtained in Example 1.

[0205] Antibacterial activity test:

[0206] The minimum inhibitory concentration (MIC) was determined using the microbroth dilution method with unmodified chlorhexidine as a control.

[0207] Test strains: Staphylococcus aureus (ATCC 25923) and Escherichia coli (ATCC 25922).

[0208] Culture conditions: 37℃, 24 hours.

[0209] Cytotoxicity test:

[0210] Cytotoxicity against human fibroblasts (L929) was evaluated using the CCK-8 assay.

[0211] Cell culture conditions: 37℃, 5% CO2, culture for 48 hours.

[0212] Set concentration gradients: 0, 50, 100, 150, 200, 250 μg / mL.

[0213] Table 6. Biological evaluation results of Example 4

[0214]

[0215] Therefore, as can be seen from Example 4:

[0216] Biological evaluation results showed that the product had excellent antibacterial activity (MIC value reduced by 50%) and good biocompatibility (IC50 > 200 μg / mL). Safety indicators such as cytotoxicity and hemolysis were superior to the control, confirming the product's safety and efficacy.

[0217] Example 5: Chlorhexidine digluconate was treated with ultrasound under the conditions of Example 1, and the dispersion effect was investigated.

[0218] Dispersion process parameters:

[0219] Ultrasonic power: 350W

[0220] Frequency: 22kHz

[0221] Intermittent mode: Work for 3 minutes / Stop for 1 minute

[0222] Total time: 18min

[0223] Temperature control: 15-20℃ water bath

[0224] Table 7. Evaluation of Dispersion Effect in Example 5

[0225]

[0226] Therefore, as can be seen from Example 5:

[0227] Ultrasonic dispersion reduced the average particle size of chlorhexidine from 5.2 μm to 2.6 μm, significantly improving dispersion stability. The zeta potential increased by 82.1%, confirming the stability of the dispersion system and laying the foundation for improved product quality.

[0228] It should be noted that all data in the above embodiments are the average values ​​of three 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 that of the prior art.

[0229] Specifically, this invention successfully solves various problems existing in the prior art through systematic process optimization and strict quality control. All product indicators are superior to the control, demonstrating significant innovation and practical value, and making it suitable for industrial production.

[0230] 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 described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing hyaluronic acid chlorhexidine having a bacteriostatic effect, characterized in that, The method comprises the following steps: S1) dissolving sodium hyaluronate in deionized water, stirring and dissolving at 40-45℃ for 2-3 hours to prepare a sodium hyaluronate solution with a mass fraction of 2-3%, and then cooling to room temperature; S2) dissolving chlorhexidine digluconate in deionized water, ultrasonic dispersion for 15-20 minutes to prepare a chlorhexidine digluconate solution with a mass fraction of 1-2%; S3) slowly adding the chlorhexidine digluconate solution in step S2 to the sodium hyaluronate solution in step S1 at a dropping rate of 2-3 mL / min at 30-35℃, while maintaining a stirring rate of 600-800 rpm, and continuing to stir for 2-4 hours after the addition is completed; S4) slowly adding anhydrous ethanol pre-cooled to 4℃ to the reaction mixture at a rate of 5-8 mL / min, and standing for 4-6 hours for precipitation; the precipitation process is carried out in the dark, S5) filtering the precipitate collected with a filter membrane with a pore size of 0.45 μm, and repeatedly washing with ethanol solutions with volume fractions of 75%, 85% and 95% in sequence for 3-5 times, each time for not less than 30 minutes; the washing process is carried out in a low-temperature and dark environment, S6) placing the obtained precipitate in a vacuum drying oven at 40-45℃, and increasing the temperature at a rate of 1℃ / min, drying for 8-12 hours, and maintaining the vacuum degree at -0.08 MPa to -0.1 MPa to obtain chlorhexidine hyaluronate, the molecular weight of the sodium hyaluronate in step S1 is 1000-1500 kDa, the residual protein content of the sodium hyaluronate is less than 0.1%, and the residual heavy metal content is less than 10 ppm, the specific conditions of the reaction in step S3 are: reaction temperature: 32±0.5℃; 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, the specific requirements of the drying process in step S6 are: S61) temperature increasing stage: increasing the temperature from room temperature to 40-45℃ at a rate of 1℃ / min; S62) constant temperature stage: maintaining at 40-45℃ for 8-10 hours; S63) temperature decreasing stage: naturally cooling to room temperature; S64) vacuum degree: gradually increasing with the temperature, and finally maintaining at -0.08 MPa to -0.1 MPa.

2. The method for preparing hyaluronic acid chlorhexidine according to claim 1, characterized in that, when preparing the sodium hyaluronate solution in step S1, one of the following stirring modes is adopted: mechanical stirring at a speed of 200-400 rpm; or magnetic stirring at a speed of 300-500 rpm; or mechanical stirring combined with magnetic stirring, wherein the mechanical stirring speed is 200-400 rpm and the magnetic stirring speed is 300-500 rpm.

3. The method for preparing hyaluronic acid chlorhexidine according to claim 1, characterized in that, the ultrasonic dispersion conditions in step S2 are: ultrasonic power: 300-400 W; ultrasonic frequency: 20-25 kHz; working mode: intermittent ultrasonic, the ratio of working time to intermittent time is 3:

1.

4. The method for preparing hyaluronic acid chlorhexidine according to claim 1, characterized in that, in step S4, the amount of anhydrous 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℃.

5. The method for preparing chlorhexidine hyaluronic acid according to claim 1, characterized in that, the washing process in step S5 comprises: S51) First washing with 75% ethanol solution for 2 times, 30 minutes each time; S52) Second washing with 85% ethanol solution for 2 times, 45 minutes each time; S53) Third washing with 95% ethanol solution for 1 time, 60 minutes.

6. The method for preparing chlorhexidine hyaluronic acid according to claim 1, characterized in that, The step S6 is followed by the following determination steps: S71) Determination of the molecular weight distribution and grafting rate of the product by gel permeation chromatography; S72) Determination of the free chlorhexidine content by high performance liquid chromatography; S73) If the determination results meet the following conditions, the product is qualified: the molecular weight distribution is 800-1200 kDa, the grafting rate is ≥ 85%, and the free chlorhexidine content is ≤ 0.5%; S74) If the above conditions are not met, the product is re-dissolved and purified by an ultrafiltration membrane with a molecular weight cut-off of 100 kDa, and the cut-off liquid is collected to repeat steps S4 to S6.

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