Method for preparing a sulfobetaine hyaluronic acid derivative and use thereof

Abstract

The application discloses a preparation method and application of a sulfonated betaine hyaluronic acid derivative, and belongs to the technical field of biological medical material preparation. The synthesis method comprises the following steps: firstly, hyaluronic acid is reacted with N,N-dimethylaminopropylamine to obtain an intermediate hyaluronic acid-dimethylaminopropylamine; and secondly, the intermediate is reacted with propenesulfonic acid lactone to obtain the sulfonated betaine hyaluronic acid derivative. The sulfonated betaine hyaluronic acid derivative has the characteristics of sulfonation, betaine and zwitterion, has the physical and chemical properties and physiological properties of moisturizing performance, antioxidation, anti-inflammation, antibiosis, anticoagulation and anti-hyaluronidase activity, has good lubricating performance and transdermal absorption performance, and has a wide application prospect in the fields of wound repair, transdermal absorption, drug delivery, cartilage / bone regeneration engineering and the like.

Description

Technical Field

[0001] This invention relates to a method for preparing a sulfonated betaine hyaluronic acid derivative and its application, belonging to the field of biomaterials technology. Background Technology

[0002] Hyaluronic acid (HA) is a natural glycosaminoglycan composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. It is widely found in human tissues, such as cartilage, skin, synovial fluid, umbilical cord, vitreous humor, and intervertebral disc nucleus. Besides its lubricating function, HA plays a crucial role in cell proliferation, migration, angiogenesis, and cell signaling. Due to its unique physicochemical properties, HA is widely used in orthopedics, ophthalmology, cosmetics, pharmaceuticals, and food.

[0003] However, the inherent degradation and instability of natural hyaluronic acid (HA) in vivo limit its applications. Since HA contains carboxyl, hydroxyl, acetamino, and reduction terminals that can be chemically modified, functionalization is a viable strategy. HA derivatization includes esterification, cross-linking, grafting, and the formation of HA-metal complexes, such as acetylated hyaluronic acid, silanized hyaluronic acid, and carboxymethyl hyaluronic acid, thereby improving its physicochemical and biological properties and greatly expanding its application areas, especially in the field of medical materials.

[0004] Glycosaminoglycans (GAGs) include four categories: HA, chondroitin sulfate (CS) / dermatan sulfate (DS), heparan sulfate (HS) / heparin (HP), and keratin sulfate (KS). From a molecular structure perspective, GAGs are divided into sulfated and non-sulfated types, with HA being the only non-sulfated GAG. Sulfated and non-sulfated GAGs exhibit different properties. On the one hand, HA is weakly negatively charged, thus it cannot complex with core proteins to form proteoglycans. On the other hand, sulfated GAGs, such as heparin, are common clinical natural anticoagulants, but their excessively high anticoagulant activity can easily cause serious adverse reactions such as bleeding and thrombocytopenia. Furthermore, the degree of sulfatedity of sulfated GAGs, to a certain extent, determines their physicochemical properties and biological activity. Existing literature reports that sulfated polysaccharide derivatives have low degrees of substitution, thus limiting their clinical application. Therefore, developing a novel, multifunctional, and highly safe sulfated GAG has significant clinical value and economic benefits. Summary of the Invention

[0005] Based on the above technical background, the purpose of this invention is to provide a method for preparing sulfonated betaine hyaluronic acid derivatives, which have good properties in terms of anti-inflammatory, anti-fouling, anticoagulant and anti-hyaluronidase, and can be applied in wound repair, transdermal absorption, drug delivery, cartilage / bone regeneration and other fields.

[0006] To achieve the objectives of this invention, the technical solution provided by this invention is as follows:

[0007] This invention provides a sulfonated betaine hyaluronic acid derivative, the structure of which is shown in Formula I below:

[0008]

[0009] Wherein, R is Na, K, Zn, or H; m and n are integers, m is between 30 and 2600, n is between 20 and 1700, and m / n is between 0.3 and 4.0. Furthermore, the sulfonated betaine hyaluronic acid derivative exists in the form of an acid salt.

[0010] The synthetic route for the above-mentioned sulfonated betaine hyaluronic acid derivative of the present invention is as follows:

[0011]

[0012] The present invention also provides a method for preparing sulfonated betaine hyaluronic acid derivatives, comprising the following steps:

[0013] S1: Hyaluronic acid and / or hyaluronic acid salt, N,N-dimethylaminopropylamine are dissolved in solvent I, then a condensing agent and a catalyst are added, and the mixture is reacted at a certain temperature for 36-48 hours to obtain reaction solution I. Then, reaction solution I is precipitated with alcohol or separated by membrane, and freeze-dried to obtain the intermediate hyaluronic acid-dimethylaminopropylamine.

[0014] S2: Hyaluronic acid-dimethylaminopropylamine is dispersed in organic solvent II, 1,3-propanesulfonic acid lactone is added, and the mixture is reacted at a certain temperature for 12-72 h to obtain reaction solution II. Then, reaction solution II is separated by alcohol precipitation or membrane separation, and freeze-dried to obtain sulfonated betaine hyaluronic acid derivative.

[0015] Furthermore, the hyaluronic acid salt mentioned in step S1 includes one or more of sodium hyaluronate, potassium hyaluronate, or zinc hyaluronate.

[0016] Furthermore, the molecular weight of the hyaluronic acid and hyaluronic acid salts mentioned in step S1 is 10 to 1000 kDa.

[0017] Preferably, the molecular weight of the hyaluronic acid and hyaluronic acid salt in step S1 is 800-1000 kDa.

[0018] Furthermore, the mass ratio of hyaluronic acid and / or hyaluronic acid salt to N,N-dimethylaminopropylamine in step S1 is 1:(1.1 to 1.5).

[0019] Preferably, the mass ratio of hyaluronic acid and / or hyaluronic acid salt to N,N-dimethylaminopropylamine in step S1 is 1:(1.1 to 1.2).

[0020] Furthermore, the solvent I mentioned in step S1 includes any one of water, formamide, or DMSO.

[0021] Preferably, the solvent I in step S1 is water.

[0022] Furthermore, in step S1, the volume ratio of solvent I to the mass of hyaluronic acid and / or hyaluronic acid salt is (10-30) mL:1 g.

[0023] Preferably, in step S1, the volume ratio of solvent I to the mass of hyaluronic acid and / or hyaluronic acid salt is (10-15) mL:1 g.

[0024] Furthermore, the condensing agent mentioned in step S1 is either 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride or N,N'-dicyclohexylcarbodiimide.

[0025] Preferably, the condensing agent in step S1 is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride.

[0026] Furthermore, in step S1, the amount of condensing agent used is 20-50% of the mass of hyaluronic acid and / or hyaluronic acid salt.

[0027] Preferably, the amount of condensing agent used in step S1 is 40-50% of the mass of hyaluronic acid and / or hyaluronic acid salt.

[0028] Furthermore, the catalyst mentioned in step S1 is any one of triethylamine, pyridine, or 4-dimethylaminopyridine.

[0029] Preferably, the catalyst in step S1 is 4-dimethylaminopyridine.

[0030] Furthermore, in step S1, the amount of catalyst used is 20-50% of the mass of hyaluronic acid and / or hyaluronic acid salt.

[0031] Preferably, the amount of catalyst used in step S1 is 40-50% of the mass of hyaluronic acid and / or hyaluronic acid salt.

[0032] Furthermore, the specific temperature mentioned in step S1 is 20–30°C.

[0033] Furthermore, in step S2, organic solvent II includes any one of ethanol, formamide, or 1,2-dichloroethane.

[0034] Preferably, the organic solvent II in step S2 is ethanol.

[0035] Furthermore, in step S2, the volume ratio of organic solvent II to the mass ratio of hyaluronic acid-dimethylaminopropylamine is (10-20) mL:1 g.

[0036] Preferably, in step S2, the volume ratio of organic solvent II to the mass ratio of hyaluronic acid-dimethylaminopropylamine is (15-20) mL:1 g.

[0037] Furthermore, in step S2, the mass ratio of 1,3-propanesulfonic acid lactone to hyaluronic acid-dimethylaminopropylamine is 1:(1.5-2).

[0038] Furthermore, in step S2, the temperature is 55–65°C.

[0039] The present invention provides a sulfonated betaine hyaluronic acid derivative prepared according to the above method.

[0040] The present invention relates to the application of sulfonated betaine hyaluronic acid derivatives in the preparation of drugs for wound repair, transdermal absorption, drug delivery, and cartilage / bone regeneration engineering.

[0041] This invention also provides a method for preparing a highly substituted sulfonated betaine hyaluronic acid derivative, comprising the following steps:

[0042] S1: Hyaluronic acid and N,N-dimethylaminopropylamine are dissolved in water, then a condensing agent and a catalyst are added, and the mixture is reacted at 20-30°C for 24-48 hours to obtain reaction solution I. Then, reaction solution I is precipitated with alcohol or separated by membrane, and freeze-dried to obtain the intermediate hyaluronic acid-dimethylaminopropylamine.

[0043] Hyaluronic acid has a molecular weight of 10 kDa;

[0044] The mass ratio of hyaluronic acid to N,N-dimethylaminopropylamine is 1:1.2;

[0045] The volume ratio of water to the mass of hyaluronic acid is 10 mL: 1 g;

[0046] The condensing agent is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride;

[0047] The amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride is 50% of the mass of hyaluronic acid;

[0048] The catalyst is 4-dimethylaminopyridine;

[0049] The amount of 4-dimethylaminopyridine used is 50% of the mass of hyaluronic acid and / or hyaluronic acid salt;

[0050] S2: Hyaluronic acid-dimethylaminopropylamine was dispersed in ethanol, 1,3-propanesulfonic acid lactone was added, and the mixture was reacted at 55-65℃ for 12-72 h to obtain reaction solution II. Then, reaction solution II was precipitated with alcohol or separated by membrane, and freeze-dried to obtain sulfonated betaine hyaluronic acid derivative.

[0051] The volume ratio of ethanol to the mass ratio of hyaluronic acid-dimethylaminopropylamine is 20 mL: 1 g.

[0052] The mass ratio of 1,3-propanesulfonic acid lactone to hyaluronic acid-dimethylaminopropylamine is 1:2.

[0053] The present invention has the following beneficial effects:

[0054] The sulfonated betaine hyaluronic acid derivative provided in this invention has stronger negative charge and higher affinity, thus exhibiting good moisturizing, antioxidant, anti-inflammatory, anticoagulant and biocompatibility properties, as well as good lubricating and transdermal absorption properties. It has broad application prospects in wound repair, transdermal absorption, drug delivery, cartilage / bone regeneration engineering and other fields. Attached Figure Description

[0055] Figure 1 The IR spectrum of sulfonated betaine hyaluronic acid SB-HA;

[0056] Figure 2 The sulfonated betaine hyaluronic acid SB-HA of Example 1 1 H NMR spectrum;

[0057] Figure 3 The sulfonated betaine hyaluronic acid SB-HA of Example 2 1 H NMR spectrum;

[0058] Figure 4 The sulfonated betaine hyaluronic acid SB-HA of Example 3 1 H NMR spectrum;

[0059] Figure 5 For Comparative Example 1, sulfonated betaine hyaluronic acid SB-HA 1 H NMR spectrum;

[0060] Figure 6 The diagram shows the moisturizing properties of HA and SB-HA.

[0061] Figure 7 Free radical scavenging rate;

[0062] Figure 8 In vitro cytotoxicity of SB-HA;

[0063] Figure 9The anti-inflammatory properties of sulfonated betaine hyaluronic acid SB-HA;

[0064] Figure 10 The anticoagulant properties of sulfonated betaine hyaluronic acid SB-HA. Detailed Implementation

[0065] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0066] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0067] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0068] Example 1

[0069] S1: Sodium hyaluronate (10 kDa, 2.0 g) and N,N-dimethylaminopropylamine (2.4 g) were dissolved in 20 mL of water, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.4 g) and 4-dimethylaminopyridine (0.4 g) were added. The mixture was reacted at 25 °C for 36 h. After the reaction was completed, the reaction solution was poured into 200 mL of ethanol, centrifuged, washed, and then freeze-dried to obtain hyaluronic acid-dimethylaminopropylamine (AHA) with a yield of 80%.

[0070] S2: 5.0 g of hyaluronic acid-dimethylaminopropylamine was dispersed in 100 mL of ethanol, and 2.5 g of 1,3-propanesulfonic acid lactone was added. The mixture was reacted at 60 °C for 12 h. After the reaction was completed, the reaction solution was poured into 300 mL of ethanol, centrifuged, washed, and then freeze-dried to obtain sulfonated betaine hyaluronic acid derivative (SB-HA) with a yield of 85%.

[0071] Example 2

[0072] The preparation process was carried out in accordance with Example 1, except that the reaction temperature in step S1 was adjusted to 40°C and the reaction time was 24 h.

[0073] Example 3

[0074] The preparation process was carried out according to Example 1, except that the amount of hyaluronic acid-dimethylaminopropylamine in step S2 was adjusted to 1g and the amount of 1,3-propanesulfonic acid lactone was adjusted to 1.8g.

[0075] Comparative Example 1

[0076] Following the preparation steps described above, the reaction order was adjusted to first synthesize sulfobetaine and then graft it onto hyaluronic acid to prepare the sulfonated betaine hyaluronic acid derivative (SB-HA). The specific steps are as follows:

[0077] S1: Acetic anhydride (25 mL) was slowly added dropwise to a solution of N,N-dimethylaminopropylamine (2.0 g) in chloroform (15 mL), and the reaction was carried out at room temperature for 6 h. After the reaction was completed, chloroform was removed by rotary evaporation, the residue was extracted with ethyl acetate, the organic phase was collected and dried with anhydrous magnesium sulfate (3.0 g), and a pale yellow oil was obtained after filtration and rotary evaporation, which is boc-protected dimethylaminopropylamine I.

[0078] S2: Intermediate I and 1,3-propanesulfonic acid lactone (4.1 g) were dissolved in DMF (50 mL) and reacted at room temperature for 10 h. After the reaction was completed, the product was filtered to obtain a white solid product. The white solid product was dissolved in methanol, and excess 10 wt% hydrochloric acid methanol solution was added dropwise. The reaction was carried out overnight. After the reaction was completed, the reaction solution was filtered and dried to obtain a white solid product, namely intermediate propanesulfonic acid dimethylaminopropylamine II.

[0079] S3: Hyaluronic acid HA (20kDa, 0.04g) was dissolved in formamide (5.0mL), DMAP (0.04g) and CDI (0.15g) were added, and the mixture was stirred at room temperature for 1h; then intermediate II (0.10g) was added, and the mixture was reacted at room temperature for 24h; after the reaction was completed, the mixture was dialyzed and freeze-dried to obtain sulfonated betaine hyaluronic acid (SB-HA).

[0080] Example 4: Structural characterization of sulfonated betaine hyaluronic acid derivatives

[0081] Analysis of the infrared spectrum of Example 1 ( Figure 1 Significant infrared absorption peaks can be observed in the product within a specific wavelength range, with these peaks located at 3100-3500 cm⁻¹. -1 1741cm -1 1558cm -1 1375cm -1 1230cm -1 and 612cm -1 Among them, 3100-3500cm -1 The absorption peak in this region corresponds to the stretching vibration of the secondary amine NH, at 1741 cm⁻¹.-1 The absorption peak is the stretching vibration peak of the C=O bond. 1558 cm⁻¹ -1 The absorption peak at 1375 cm⁻¹ corresponds to the bending vibration of amide NH (amide II band), which usually overlaps with the amide I band. -1 The absorption peak at 1230 cm⁻¹ is attributed to the asymmetric stretching vibration of sulfonyl SO₃. -1 It is the symmetric stretching vibration peak of sulfonyl SO3, and 612 cm⁻¹ is the peak of the symmetric stretching vibration of sulfonyl SO₃. -1 The absorption peak at the specified location corresponds to the out-of-plane bending vibration of amide NH (amide IV band), indicating that the structure is correct. The characteristic CH peak on the HA sugar ring in Examples 1-3 appears in the NMR region of δ = 3.2–4.7 ppm. The characteristic peak of the methyl group linked to the amide group appears in the region of δ = 1.9 ppm–2.1 ppm. Analysis of the integrated area confirmed that δ = 3.34 ppm is the characteristic peak of the glucuronic acid unit H-2, indicating one proton, while δ = 3.59 ppm is the characteristic peak of the adjacent H-3, indicating the presence of two protons. The characteristic peaks of SB-HA and AHA samples in the region of δ = 1.9 ppm–2.1 ppm remain well-preserved, without significant chemical shifts to higher or lower fields, indicating that the acetyl group of HA is not destroyed.

[0082] Based on the integral calculation of the spectral data, the grafting rate of SB-HA in Example 1 was 48%, that in Example 2 was 18%, that in Example 3 was 31%, and that in Comparative Example 1 was 8%. Compared with Examples 2 and 3 and Comparative Example 1, the grafting rate comparison showed that Example 1 was the preferred condition.

[0083] Example 5: Moisturizing performance

[0084] An in vitro weighing method was used. The temperature and humidity of the constant temperature and humidity chamber were set at 28℃ and 50% and stabilized for 24 hours. Then, culture dishes of the same area were weighed on an electronic balance to obtain M1. 50 mg of H2O, HA, and SB-HA prepared in Example 1 were weighed into each culture dish, dissolved in 10 mL of deionized water, and weighed to obtain M2. The culture dishes were then placed in the stable constant temperature and humidity chamber, and removed and weighed at regular intervals (30 min, 1 h, 2 h, 3 h, 5 h, 8 h, 12 h, 24 h) to obtain M3. The moisture retention rate was calculated as follows: Moisture retention rate = [(M3 - M1) / (M2 - M1)] × 100%.

[0085] Experimental results show that under conditions of 28℃ and 50% humidity, the moisture retention rates of the two products after 24 hours reached 95.38% (HA) and 95.47% (SB-HA), respectively. Both products exhibit good moisturizing effects, with SB-HA showing slightly better moisturizing performance than HA.

[0086] Example 6: Antioxidant properties

[0087] S1: Dissolve 4.8 mg (accurately weighed) of DPPH reagent in anhydrous ethanol and quantitatively transfer it into a 100 mL volumetric flask. Dilute to the mark with anhydrous ethanol and shake well to obtain a DPPH stock solution with a concentration of 0.012 mmol / L. Then store it in a refrigerator for later use.

[0088] S2: Accurately weigh 50 mg of SB-HA sample, 50 mg of HA, and 50 mg of vitamin C prepared in Example 1, dissolve them in deionized water, and quantitatively transfer them to 10 mL volumetric flasks. Dilute these solutions to prepare sample solutions of 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, and 0.5 mg / mL, respectively. Add 3 mL of DPPH stock solution and 1 mL of sample solution to 10 mL colorimetric tubes, shake well, let stand, and react in the dark for 30 min. Measure the absorbance (A) at 517 nm. Use the corresponding mixture without sample as a blank control and record the corresponding Ai, Aj, and Ac values.

[0089] S3: The DPPH free radical scavenging rate of the sample was calculated using the formula: K(%) = [1 - (Ai - Aj) / Ac] × 100%

[0090] In the formula: Ai is the absorbance value of the sample after reacting with DPPH (1 mL sample solution + 3 mL DPPH solution), Aj is the absorbance value of the blank sample (1 mL sample solution + 3 mL anhydrous ethanol solvent), and Ac is the absorbance value of DPPH without sample (1 mL blank solvent + 3 mL DPPH solution).

[0091] The experimental results are shown in Figure 7, IC 50 (HA) > IC 50 (SB-HA), which further indicates that SB-HA has better antioxidant capacity than HA.

[0092] Example 7: Biocompatibility

[0093] Fibroblasts were charged at 3.5 × 10 3Cells were seeded at a density of 10 cells / well into 96-well plates and incubated overnight in an incubator (37°C, 5% CO2). Sulfonated betaine hyaluronic acid solutions (Example 1) were prepared at concentrations of 6 mg / mL, 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.1 mg / mL. Each plate was used as a blank control group and groups with concentrations of 6 mg / mL, 5 mg / mL, 4 mg / mL, 3 mg / mL, 2 mg / mL, 1 mg / mL, 0.5 mg / mL, and 0.1 mg / mL. When the cell deposition rate in the 96-well plates reached 20%–30%, the culture medium was removed, and different concentrations of sulfonated betaine hyaluronic acid were added to each well according to the groupings, with 200 μL added per well and 3 replicates per group. The plates were then incubated for 24 hours. Cell viability was assessed using the MTT assay. After culturing the cells for 24 hours, add MTT solution and incubate in the dark. After 4 hours, aspirate the solution and add DMSO to each well. Shake the plate on a microplate reader and set the absorbance of the microplate reader to 490 nm, then take the reading.

[0094] Experimental results are as follows Figure 8 As shown, under culture conditions with different concentrations of SB-HA solution, cell viability remained above 80%, indicating that SB-HA has good biocompatibility.

[0095] Example 8: Anti-inflammatory properties

[0096] THP-1 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum at 37°C and 50 mL / L CO2. M0 macrophages were induced to differentiate using 100 nmol / L phorbol ester. M1 macrophages were induced using serum-free medium containing 1 μg / mL LPS and 20 ng / mL INF-γ. Simultaneously, SB-HA prepared in Example 1 was dissolved in the medium to a concentration of 3 mg / mL, serving as the intervention group. After 24 h of culture, samples were collected for subsequent experiments.

[0097] Total RNA was extracted from cells using the Trizol reagent kit, and... The All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) kit series (Transgen, China) was used to reverse transcribe RNA into cDNA. The reaction conditions were 42℃ for 15 min and 85℃ for 5 s, respectively, to amplify TNF-α and IL-6 cDNA samples. Real-time quantitative PCR was used to detect the mRNA expression of relevant inflammatory factors. The RT-PCR reaction conditions and system were set according to the PerfectStart Green qPCR SuperMix kit series (Transgen, China), with GAPDH (Sangon, China) as an internal control. -ΔΔCT The relative expression levels of TNF-α and IL-6 mRNA were calculated using a method.

[0098] Studies have shown that, compared with the blank control group, the TNF-α and IL-6 clearance rates in the SB-HA intervention group and the inflammation model group were significantly improved. SB-HA inhibited the expression of inflammation-related factors, demonstrating its good anti-inflammatory effect. The experimental results are as follows: Figure 9 As shown.

[0099] Example 8: Anticoagulant properties

[0100] To assess the hemolysis rate of the biomaterials, sterile defibrinated sheep blood (Shigouyi, China) was centrifuged at 3000 rpm for 15 min to obtain blood cells. SB-HA (3 mg) prepared in Example 1 was dissolved in 1 mL of PBS solution. These solutions were then mixed with 20 μL of blood cells; after incubation at 37°C for 4 h, the mixture was centrifuged at 3000 rpm for 15 min. The samples were placed horizontally, and the hemolysis phenomenon was photographed. Next, the supernatant of the samples was aspirated and analyzed using a microplate reader (ELISA reader). The absorbance of the sample was measured at a wavelength of 542 nm using a 200PRO (TECAN, Switzerland) microscope. The experiment was repeated three times.

[0101] Experimental results are as follows Figure 10 As shown, the Triton X-100 group is red, due to the rupture of red blood cells, while the supernatant of the SB-HA group is colorless, indicating that hemolysis has not occurred. Absorbance readings on a microplate reader show that the hemolysis rate of the SB-HA group is less than 5%, indicating that the material is blood-compatible.

[0102] Example 9: Antibacterial properties

[0103] SB-HA solutions of 15 mg / mL, 10 mg / mL, and 5 mg / mL (Example 1) were prepared and stored at 4°C to promote dissolution. Next, 100 μL of bacterial suspension was added to a solid culture medium, and a spreading method was used to ensure uniform distribution of the bacterial suspension on the surface of the medium. Several filter paper discs were prepared using a 6 mm diameter punch and sterilized by autoclaving and drying to eliminate potential contamination. These filter paper discs were then immersed in different concentrations of the SB-HA solution prepared in Example 1 for 20 min to ensure sufficient adsorption, and then evenly distributed onto the culture medium inoculated with the bacterial suspension using tweezers. Each concentration treatment was repeated three times on each culture medium as a parallel experiment, with filter paper discs soaked in 75% ethanol as a positive control and filter paper discs soaked in sterile water as a negative control. The treated culture dishes were incubated with bacteria at 37°C for 24 h. After the experiment, the diameter of the inhibition zone was measured using calipers, and the average value was calculated to quantitatively evaluate the antibacterial effect of SB-HA.

[0104] To determine the minimum inhibitory concentration (MIC) of SB-HA, this invention employed a twofold dilution method in test tubes. First, a series of SB-HA solutions with varying concentrations were prepared from the roots: 10 mg / mL, 5 mg / mL, 2.5 mg / mL, 1.25 mg / mL, 0.625 mg / mL, 0.312 mg / mL, 0.156 mg / mL, 0.078 mg / mL, 0.039 mg / mL, 0.019 mg / mL, and 0.009 mg / mL. Next, 5 mL of each of the different concentrations of HA solution was added to 11 sterile test tubes, followed by 0.5 mL of bacterial suspension and 5 mL of the corresponding sterile liquid culture medium in each tube. As controls, tube 0 (negative control) contained 5.5 mL of sterile water and 5 mL of liquid culture medium, while tube 12 (positive control) contained 5 mL of sterile water, 5 mL of liquid culture medium, and 0.5 mL of bacterial suspension. The treated samples were cultured separately according to the culture conditions for their respective bacterial strains, with a time range of 24 hours. After the culture was completed, the growth was assessed by observing the turbidity in the test tubes.

[0105] The experimental results are shown in Tables 1 and 2. SB-HA exhibits a certain concentration dependence, and its antibacterial effect may increase with increasing SB-HA concentration. At low concentrations, sulfated HA may only show slight antibacterial activity, while at high concentrations, it shows a good antibacterial effect.

[0106] Table 1. MIC determination results of SB-HA

[0107]

[0108]

[0109] Note: "—" indicates no bacterial growth; "+" indicates a small amount of bacterial growth; "++" indicates a large amount of bacterial growth.

[0110] Table 2 shows the MIC determination results of SB-HA.

[0111]

[0112] Note: Different lowercase superscript letters in the same row indicate significant differences (p < 0.05); different uppercase superscript letters in the same column indicate significant differences (p < 0.05).

[0113] The embodiments provided above are not intended to limit the scope of the invention, nor are the described steps intended to limit the order of execution. Any obvious modifications made to the invention by those skilled in the art based on existing common knowledge also fall within the scope of protection defined by the claims.

Claims

1. A sulfonated betaine hyaluronic acid derivative, characterized in that, The structure is as shown in Equation I below: Formula I Where R is Na, K, Zn or H; m and n are integers, m is 30~2600, n is 20~1700, and m / n is 0.3~4.

0.

2. The method for preparing the sulfonated betaine hyaluronic acid derivative according to claim 1, characterized in that, Includes the following steps: S1: Hyaluronic acid and / or hyaluronic acid salt, N,N-dimethylaminopropylamine are dissolved in solvent I, then a condensing agent and a catalyst are added, and the mixture is reacted at a certain temperature for 36-48 h to obtain reaction solution I. Then, reaction solution I is precipitated with alcohol or separated by membrane, and freeze-dried to obtain intermediate hyaluronic acid-dimethylaminopropylamine. S2: Hyaluronic acid-dimethylaminopropylamine is dispersed in organic solvent II, 1,3-propanesulfonic acid lactone is added, and the mixture is reacted at a certain temperature for 12-72 h to obtain reaction solution II. Then, reaction solution II is separated by alcohol precipitation or membrane separation, and freeze-dried to obtain sulfonated betaine hyaluronic acid derivative.

3. The method according to claim 2, characterized in that, In step S1, the hyaluronic acid salt includes one or more of sodium hyaluronate, potassium hyaluronate, or zinc hyaluronate; the molecular weight of the hyaluronic acid and the hyaluronic acid salt is 10~1000 kDa.

4. The method according to claim 2, characterized in that, The mass ratio of hyaluronic acid and / or hyaluronic acid salt to N,N-dimethylaminopropylamine in step S1 is 1:(1.1~1.5).

5. The method according to claim 2, characterized in that, The solvent I mentioned in step S1 includes any one of water, formamide or DMSO; the volume ratio of solvent I to the mass of hyaluronic acid and / or hyaluronic acid salt is (10~30) mL : 1 g.

6. The method according to claim 2, characterized in that, In step S1, the condensing agent is any one of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N,N'-dicyclohexylcarbodiimide; the amount of condensing agent used is 20-50% of the mass of hyaluronic acid and / or hyaluronic acid salt; the catalyst is any one of triethylamine, pyridine, or 4-dimethylaminopyridine; the amount of catalyst used is 20-50% of the mass of hyaluronic acid and / or hyaluronic acid salt.

7. The method according to claim 2, characterized in that, The specific temperature mentioned in step S1 is 20~30℃.

8. The method according to claim 2, characterized in that, In step S2, organic solvent II includes any one of ethanol, formamide, or 1,2-dichloroethane; the volume ratio of organic solvent II to the mass ratio of hyaluronic acid-dimethylaminopropylamine is (10~20) mL : 1 g.

9. The method according to claim 2, characterized in that, In step S2, the mass ratio of 1,3-propanesulfonic acid lactone to hyaluronic acid-dimethylaminopropylamine is 1:(1.5~2), and the specified temperature is 55~65℃.

10. The use of the sulfonated betaine hyaluronic acid derivative as described in claim 1, or the sulfonated betaine hyaluronic acid derivative prepared by the method described in any one of claims 2 to 9, in the preparation of drugs for wound repair, transdermal absorption, drug delivery, and cartilage / bone regeneration engineering.

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