Quaternary ammonium salt modified hyaluronic acid as well as preparation method and application thereof
By shielding the positive charge of quaternary ammonium salts in hyaluronic acid and expressing the bactericidal properties after degradation by hyaluronidase, the cytotoxicity problem of quaternary ammonium salts is solved, and enzyme-sensitive antibacterial properties and efficient bactericidal effects are achieved at the site of bacterial infection.
Patent Information
- Application Number
- CN202510714730.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-30
- Publication Date
- 2025-10-17
AI Technical Summary
Although quaternary ammonium salts have excellent bactericidal properties, the cytotoxicity caused by their positive charge is harmful to human cells. Existing technologies make it difficult to reduce their cytotoxicity without reducing their bactericidal properties.
The positive charge of the quaternary ammonium salt is shielded by the carboxyl group of hyaluronic acid, and the bactericidal property is expressed after degradation at the bacterial infection site by hyaluronidase. The hyaluronic acid modified with the quaternary ammonium salt has a molecular formula of X=4-10, Y=7-20, and a molar ratio of N to M of 2.0-1.0:1. The preparation method includes adding triethylamine and 4,6-trichlorobenzoyl chloride to a tetrahydrofuran solution, dialyzing and freeze-drying after the reaction, and modifying it on the surface of a medical implant.
The cytotoxicity of quaternary ammonium salt-modified hyaluronic acid was reduced under normal physiological conditions, and the bactericidal properties were expressed through enzyme sensitivity at the site of bacterial infection, thereby improving the blood compatibility and bactericidal effect of the material.
Smart Images
Figure CN120795199A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological polymer synthesis, in particular to a hyaluronic acid modified by quaternary ammonium salt, which is simple in synthesis process, suitable for industrial production, can shield the positive charge of quaternary ammonium salt through the carboxyl group of hyaluronic acid, and further reduce the cytotoxicity caused by the modification of quaternary ammonium salt, and a preparation method and application thereof. BACKGROUND
[0002] Quaternary ammonium salt has excellent bactericidal performance, which can destroy the integrity of bacteria by destroying the cell membrane and cell wall of bacteria through positive charge and hydrophobic carbon chain, so that the intracellular matrix flows out and the bacteria die. However, this bactericidal mode of quaternary ammonium salt inevitably brings cytotoxicity, which can destroy the cell membrane of normal human cells while killing bacteria. Therefore, it is important to reduce the cytotoxicity of quaternary ammonium salt.
[0003] Since the positive charge of quaternary ammonium salt is an important cause of toxicity, shielding the positive charge of quaternary ammonium salt with negative charge can effectively reduce the cytotoxicity of quaternary ammonium salt. However, this method inevitably reduces the bactericidal performance of quaternary ammonium salt, and innovative molecular structure design is needed to solve this contradiction.
[0004] In the microenvironment of bacterial infection, bacteria can secrete a large amount of hyaluronidase to degrade the tissue at the site of bacterial parasitism. Therefore, it is of important research and application value to design an antibacterial molecule which does not have bactericidal performance in normal physiological environment, but expresses bactericidal performance after being degraded by hyaluronidase at the site of bacterial infection, and has enzyme-sensitive bactericidal performance. SUMMARY
[0005] The present application proposes a hyaluronic acid modified by quaternary ammonium salt, which is simple in synthesis process, suitable for industrial production, can shield the positive charge of quaternary ammonium salt through the carboxyl group of hyaluronic acid, and further reduce the cytotoxicity caused by the modification of quaternary ammonium salt, and a preparation method and application thereof.
[0006] The present application achieves the above-mentioned purposes by the following measures:
[0007] A hyaluronic acid modified by quaternary ammonium salt, characterized in that the molecular formula is: wherein X is an integer of 4-10, Y is an integer of 7-20, and the molar ratio of N and M is 2.0-1.0:1.
[0008] In the present application, the value range of X is further preferably 4-6, the value range of Y is further preferably 7-11, and the molar ratio of N and M is further preferably 1.67:1.
[0009] The application also provides a preparation method of the hyaluronic acid modified by the quaternary ammonium salt, and is characterized in that: the quaternary ammonium salt molecules are dissolved in a tetrahydrofuran solution, then triethylamine and 4,6-trichlorobenzoyl chloride (TCBC) are added, stirring is carried out at room temperature, then the solution is added into a solution of the hyaluronic acid, reaction is carried out at room temperature, and then dialysis and freeze-drying are carried out to obtain the hyaluronic acid modified by the quaternary ammonium salt.
[0010] The solution of the hyaluronic acid in the application is obtained by the following preparation method: the hyaluronic acid is dissolved in pure water, then tetrahydrofuran, triethylamine and 4-dimethylaminopyridine are added.
[0011] In the application, the molar ratio of the quaternary ammonium salt molecules, triethylamine and 4,6-trichlorobenzoyl chloride (TCBC) is 1:1.0-3.0:1.0-2.0, and is further preferably 1:1.5:1.2.
[0012] In the application, the molar ratio of the primary hydroxyl group on the hyaluronic acid, triethylamine and 4-dimethylaminopyridine is 1:1.0-3.0:0.01-0.1, and is preferably 1:1.5:0.02.
[0013] The quaternary ammonium salt in the application has the following molecular structure: In the application, the value of X is an integer of 4-10, and is preferably 4-6, and the value of Y is an integer of 7-20, and is preferably 7-11; further, the molecular weight of the hyaluronic acid used in the application is 100,000-1,000,000, and is preferably 1,000,000-3,000,000.
[0014] The hyaluronic acid modified by the quaternary ammonium salt in the application does not have antibacterial performance before being degraded by hyaluronidase, and has antibacterial performance after being degraded by hyaluronidase, and thus has enzyme-sensitive antibacterial performance, and based on this, the application also provides an application of the hyaluronic acid modified by the quaternary ammonium salt, and is characterized in that: the hyaluronic acid modified by the quaternary ammonium salt is modified on the surface of a medical implant by a chemical grafting method or layer-by-layer self-assembly or spraying, and the medical implant includes a suture, a medical catheter, a bone nail, a bone plate and a wound dressing.
[0015] The present application grafts the quaternary ammonium salt on the hyaluronic acid molecular chain by the reaction of the primary hydroxyl group of the hyaluronic acid with the carboxyl group of the quaternary ammonium salt, retains the carboxyl group of the hyaluronic acid, controls the amount of the quaternary ammonium salt, so that the carboxyl group of the hyaluronic acid can shield the positive charge of the quaternary ammonium salt, reduces the cytotoxicity caused by the quaternary ammonium salt modification, and at the same time, in the microenvironment of bacterial infection, the hyaluronidase degrades the hyaluronic acid modified by the quaternary ammonium salt, and expresses the bactericidal performance, thereby obtaining a quaternary ammonium salt modified hyaluronic acid with enzyme sensitive antibacterial performance, specifically, before being degraded by the enzyme, the amount of the negative charge of the quaternary ammonium salt modified hyaluronic acid molecule as a whole is greater than that of the positive charge, so the bactericidal performance is not exhibited, after being degraded by the enzyme, the fragments of the quaternary ammonium salt modified hyaluronic acid with low molecular weight will have the amount of the positive charge greater than that of the negative charge, and such fragments will exhibit the bactericidal performance, while the fragments with the amount of the positive charge less than or equal to that of the negative charge will not have the bactericidal performance, and will not affect the bactericidal performance of the fragments with the amount of the positive charge greater than that of the negative charge. BRIEF DESCRIPTION OF DRAWINGS
[0016] FIG. 1 is a 1H NMR spectrum of the quaternary ammonium salt modified hyaluronic acid in Example 1 of the present application. Figure 1 FIG. 2 is a histogram of the hemolysis rate experiment of the product in the present application.
[0017] Figure 2 FIG. 3 is the bactericidal experiment results of the product in the present application.
[0018] FIG. 4 is a 1H NMR spectrum of the quaternary ammonium salt modified hyaluronic acid in Example 2 of the present application. Figure 3 FIG. 5 is a histogram of the hemolysis rate experiment of the product in Example 2 of the present application.
[0019] The present application will be further described below in combination with the drawings and examples.
[0020] Example 1
[0021] The present example provides a preparation method of quaternary ammonium salt modified hyaluronic acid, which specifically comprises: dissolving 1 gram of 100 million molecular weight hyaluronic acid (containing 2.6 mmol of primary hydroxyl group) in 100 milliliters of pure water solution, adding 30 milliliters of tetrahydrofuran, adding triethylamine (3 mmol), and 4-dimethylaminopyridine (0.026 mmol). After stirring and dissolving, a hyaluronic acid solution is obtained.
[0022] The quaternary ammonium salt (2 mmol) is dissolved in a tetrahydrofuran solution, then triethylamine (3 mmol) and 4,6-trichlorobenzoyl chloride (TCBC) (2.4 mmol) are added, stirred at room temperature for 3 hours, then added to the above hyaluronic acid solution, stirred for 24 hours, dialyzed with a dialysis bag with a molecular weight cutoff of 1000, and then freeze-dried to obtain quaternary ammonium salt modified hyaluronic acid molecule 1. 0.6 grams of the hyaluronic acid molecule 1 is dissolved in 1 mL of pure water to obtain a hyaluronic acid molecule solution 1.
[0023] The quaternary ammonium salt used in this example has the following formula:
[0024]
[0025] The quaternary ammonium salt modified hyaluronic acid obtained in this example has the following formula:
[0026]
[0027] Example 2
[0028] This example provides a method for preparing quaternary ammonium salt modified hyaluronic acid, which specifically comprises: dissolving 1 gram of 100,000 molecular weight hyaluronic acid (containing 2.6 mmol of primary hydroxyl groups) in 100 milliliters of pure water solution, adding 30 milliliters of tetrahydrofuran, adding triethylamine (3 mmol), 4-dimethylaminopyridine (0.026 mmol), and stirring to dissolve to obtain a hyaluronic acid solution;
[0029] Dissolve the quaternary ammonium salt (1 mmol) in tetrahydrofuran solution, then add triethylamine (3 mmol), 4,6-trichlorobenzoyl chloride (TCBC) (2.4 mmol), stir at room temperature for 3 hours, then add to the above hyaluronic acid solution, stir for 24 hours, dialyze with a dialysis bag with a molecular weight cutoff of 1000, and then lyophilize to obtain quaternary ammonium salt modified hyaluronic acid molecule 2. Dissolve 0.6 grams of hyaluronic acid molecule 2 in 1 mL of pure water to obtain hyaluronic acid molecule solution 2. The quaternary ammonium salt used in this example has the following formula:
[0030]
[0031] The quaternary ammonium salt modified hyaluronic acid obtained in this example has the following formula:
[0032]
[0033] Example 3
[0034] Dissolve 0.6 grams of hyaluronic acid molecule 2 obtained in Example 2 in 0.5 milliliters of pure water to prepare a hyaluronic acid molecule solution. Dissolve 0.33 milligrams of hyaluronidase (300 u / mg) in 50 milliliters of pure water to prepare a hyaluronidase solution. Mix the hyaluronic acid molecule solution and the hyaluronidase solution in a 1:1 ratio, shake at 37°C for 24 hours on a shaker, and obtain hyaluronic acid molecule solution 3.
[0035] The products obtained in Examples 1, 2, and 3 are evaluated for enzyme responsiveness through a hemolysis experiment. The experimental steps are as follows:
[0036] Take 10% rabbit blood plasma, respectively, with normal saline (negative control), hyaluronic acid molecule solution 1, hyaluronic acid molecule solution 2, hyaluronic acid molecule solution 3, pure water (positive control) 1:1 ratio, 37℃ shaking incubation for 90 minutes, centrifugal take the upper clear liquid, enzyme mark instrument at 540nm wavelength determination absorbance (A s ).
[0037] The hemolysis rate is calculated as follows:
[0038]
[0039] In the formula: HR represents the hemolysis rate, A 阴 represents the negative control absorbance, A 阳 represents the positive control absorbance, A s代表 corresponding experimental group solution absorbance;
[0040] As Figure 2 shown, the hemolysis rate of hyaluronic acid molecule 1 is higher, because the quaternary ammonium salt grafting rate of hyaluronic acid molecule 1 is higher, the whole molecule still shows positive charge before degradation, so the blood compatibility of hyaluronic acid molecule 1 is poor, the hemolysis rate of hyaluronic acid molecule 2 is lower, and the blood compatibility is better, because the quaternary ammonium salt grafting rate of hyaluronic acid molecule 2 is appropriate, and the charge neutralization effect of hyaluronic acid is achieved, which improves the blood compatibility of the material, and the hemolysis rate of hyaluronic acid molecule solution 3 is higher, which shows that the hyaluronic acid molecule 2 is degraded by hyaluronidase, and the positively charged molecular fragments are formed in the hyaluronic acid molecule solution 3, which proves the enzyme responsiveness of hyaluronic acid molecule 2.
[0041] The bactericidal effect of the products obtained in examples 1 and 2 is evaluated by bactericidal experiment, as follows:
[0042] Material preparation: weigh 1g of hyaluronic acid with molecular weight of 100000 without treatment and dissolve in 100ml of pure water solution, marked as control solution, and take 100ml of hyaluronic acid molecule solution 1 and 100ml of hyaluronic acid molecule solution 2, irradiation sterilization.
[0043] Bactericidal test: take staphylococcus aureus liquid grown to logarithmic phase, dilute with PBS solution to 2*10 6 cfu / mL. Take the control solution, hyaluronic acid molecule solution 1 and hyaluronic acid molecule solution 2 after irradiation sterilization, respectively, add 100ml of diluted bacteria solution, cultivate in incubator for 1 day, take 1ml of culture solution and inoculate on solid medium, cultivate in incubator for 1 day, and take photos for counting.
[0044] The results are shown in the following table: Figure 3As shown, the hyaluronic acid has no obvious sterilization effect. The modified hyaluronic acid molecules 1 and 2 have sterilization effect, because after the modified hyaluronic acid molecules are degraded by the hyaluronidase secreted by the bacteria, the fragments of the low molecular weight of the quaternary ammonium salt modified hyaluronic acid will have more positive charges than negative charges, and such fragments will exhibit sterilization performance.
Claims
1. A quaternary ammonium salt-modified hyaluronic acid, characterized in that: The molecular formula is: Wherein, X is an integer of 4-10, Y is an integer of 7-20, and the molar ratio of N to M is 2.0-1.0:
1.
2. A quaternary ammonium salt-modified hyaluronic acid according to claim 1, characterized in that, The value range of X is 4-6, the value range of Y is 7-11, and the molar ratio of N to M is 1.67:
1.
3. A method for preparing quaternary ammonium salt-modified hyaluronic acid as claimed in any one of claims 1 or 2, characterized in that: The quaternary ammonium salt molecules are dissolved in tetrahydrofuran solution, and then triethylamine and 4,6-trichlorobenzoyl chloride (TCBC) are added and stirred at room temperature. Then, the solution is added to the hyaluronic acid solution. After reaction at room temperature, the solution is dialyzed and freeze-dried to obtain quaternary ammonium salt-modified hyaluronic acid.
4. The method for preparing quaternary ammonium salt-modified hyaluronic acid according to claim 3, wherein The hyaluronic acid solution is prepared by the following method: dissolving hyaluronic acid in pure water, and adding tetrahydrofuran, triethylamine, and 4-dimethylaminopyridine.
5. The method for preparing quaternary ammonium salt-modified hyaluronic acid according to claim 3, wherein The molar ratio of the quaternary ammonium salt molecule to triethylamine and 4,6-trichlorobenzoyl chloride TCBC is 1:1.0-3.0:1.0-2.
0.
6. The method for preparing quaternary ammonium salt-modified hyaluronic acid according to claim 5, wherein The molar ratio of the quaternary ammonium salt molecule to triethylamine and 4,6-trichlorobenzoyl chloride TCBC is 1:1.5:1.
2.
7. The method for preparing quaternary ammonium salt-modified hyaluronic acid according to claim 3, wherein The molar ratio of primary hydroxyl groups on hyaluronic acid to triethylamine and 4-dimethylaminopyridine is 1:1.0-3.0:0.01-0.
1. The quaternary ammonium salt has the following molecular structure: The molecular weight of the hyaluronic acid used is 10,000-1,000,000.
8. The method for preparing quaternary ammonium salt-modified hyaluronic acid according to claim 7, wherein The molar ratio of the primary hydroxyl group on the hyaluronic acid to triethylamine and 4-dimethylaminopyridine is 1:1.5:0.
02.
9. The method for preparing quaternary ammonium salt-modified hyaluronic acid according to claim 7, wherein The molecular weight of the hyaluronic acid used is 100,000-300,000.
10. A use of the quaternary ammonium salt-modified hyaluronic acid according to any one of claims 1 or 2, characterized in that: The quaternary ammonium salt-modified hyaluronic acid is modified on the surface of a medical implant by chemical grafting, layer-by-layer self-assembly, or spraying. The medical implant includes sutures, medical catheters, bone nails, bone plates, and wound dressings.