Modified hyaluronic acid antibacterial material, antibacterial hydrogel and preparation method and application thereof

Abstract

The application relates to a modified hyaluronic acid antibacterial material, an antibacterial hydrogel and a preparation method and application thereof, relates to the technical field of antibacterial materials, and solves the technical problems that the existing hyaluronic acid-based antibacterial material has weak antibacterial activity, narrow antibacterial spectrum, is easy to rely on oxidation modification, and the hyaluronic acid molecular structure and biological activity are damaged, and the existing nano-silver antibacterial dressing has biological toxicity and poor cell compatibility, and the existing technology is difficult to simultaneously consider the broad-spectrum antibacterial performance, thermal stability, biological compatibility and wound repair capacity. The hyaluronic acid is grafted with quaternary ammonium salt with different quaternary ammonium salt alkyl chain lengths and quaternary ammonium degrees, a hyaluronic acid electrostatic grafting quaternary ammonium salt antibacterial material is prepared, and further crosslinking reaction is carried out on the antibacterial material, a crosslinking agent and glacial acetic acid to obtain an antibacterial hydrogel which has high efficient antibacterial activity, excellent biological compatibility and low cytotoxicity. The application can be applied to the field of hydrogel dressings.

Description

Technical Field

[0001] This invention relates to the field of antibacterial materials technology, specifically to a modified hyaluronic acid antibacterial material, an antibacterial hydrogel, its preparation method, and its application. Background Technology

[0002] Wound infections include common types such as postoperative wound infections, burn wound infections, diabetic wound infections, and acne wound infections. These wound infections not only have a slow healing process, but severe infections can also easily trigger systemic inflammatory responses, which can endanger the patient's life in severe cases. In clinical practice, antibiotics are the mainstream treatment for wound infections, but the overuse and improper use of antibiotics have significantly accelerated the proliferation of drug-resistant bacteria and even multidrug-resistant strains. Meanwhile, the development cycle of new antibiotics is long and the iteration speed is slow, making it difficult to meet the urgent clinical needs to combat drug-resistant bacteria.

[0003] In recent years, antibacterial hydrogel dressings, with their three-dimensional network structure similar to human soft tissue and excellent moisturizing and breathability, have become a research hotspot in wound repair, effectively covering wounds and absorbing wound exudate. These antibacterial hydrogel dressings follow the principle of moist wound healing, creating a moist and sealed repair microenvironment for the wound, accelerating tissue regeneration and healing, while physically blocking external pathogens and reducing the risk of secondary infection. Compared to traditional antibiotic administration, antibacterial hydrogel dressings avoid the risk of bacterial resistance induction while simultaneously promoting wound tissue repair. Current technologies often use nano-silver-loaded hydrogels as antibacterial wound dressings; however, high doping levels of nano-silver or long-term in vitro and in vivo application can easily lead to silver ion accumulation, posing biotoxicity risks and poor cell compatibility. Therefore, developing novel antibacterial materials with both high antibacterial properties and excellent biocompatibility to prepare medical antibacterial hydrogel dressings has significant clinical implications for wound infection control and tissue repair.

[0004] Existing antibacterial materials largely focus on natural polysaccharide biopolymers. Among them, hyaluronic acid (HA), as a natural acidic mucopolysaccharide, possesses excellent moisturizing properties, wound repair capabilities, and good biocompatibility. It can bind to receptors on the surface of skin cells, promoting fibroblast proliferation and collagen synthesis, thus creating a favorable microenvironment for wound healing. However, natural hyaluronic acid has relatively weak antibacterial activity and a narrow antibacterial spectrum, making it difficult to meet the antibacterial requirements of medical antibacterial dressings on its own, thus limiting its direct application in antibacterial wound materials. To address these issues, current research has focused on antibacterial modification of hyaluronic acid. For example, the International Journal of Biological Macromolecules published an English article entitled "Amphiphilicionic complexes of hyaluronic acid with organophosphonium compounds and their antimicrobial activity," which disclosed the synthesis of an amphoteric complex from hyaluronic acid and alkyltrimethylphosphonium compounds with different alkyl chain lengths. This complex has excellent heat resistance and thermal stability, but it still has the problem of a narrow antibacterial spectrum, showing good antibacterial effect only against Gram-positive bacteria and almost no inhibitory activity against Gram-negative bacteria. In addition, Chinese invention patent CN118812877A discloses a long-lasting antibacterial hydrogel containing polyguanidine polymers, which improves the antibacterial performance of the system by performing Schiff base chemical cross-linking reaction between oxidized hyaluronic acid and the amino groups of chitosan derivatives / polyguanidine polymers; however, this method requires oxidative modification of hyaluronic acid, which can easily cause polysaccharide molecular chain breakage, destroy the intrinsic structure and physiological activity of hyaluronic acid, and the degree of oxidation is difficult to control precisely, which can easily lead to over-oxidation, further affecting the biocompatibility of the material and the wound repair efficacy.

[0005] Therefore, developing a modified hyaluronic acid antibacterial material that does not require strong oxidative modification, has a broad antibacterial spectrum, excellent thermal stability, and good biocompatibility, and constructing a matching antibacterial hydrogel system, is of great scientific research value and practical clinical significance for expanding the clinical application of hyaluronic acid in medical wound dressings and anti-infection repair materials. Summary of the Invention

[0006] To address the challenges of existing hyaluronic acid-based antibacterial materials exhibiting weak antibacterial activity, a narrow antibacterial spectrum, and susceptibility to oxidative modification leading to damage to the hyaluronic acid molecular structure and bioactivity, as well as the biotoxicity and poor cell compatibility of existing nano-silver antibacterial dressings, current technologies struggle to simultaneously achieve broad-spectrum antibacterial performance, thermal stability, biocompatibility, and wound healing capabilities. This invention proposes a modified hyaluronic acid antibacterial material, an antibacterial hydrogel, its preparation method, and its applications. The technical solution of this invention is as follows: A modified hyaluronic acid antibacterial material, with the structural formula shown in Formula I: Formula I; In the formula, n is 10~6000; x / n×100%=1%~99%; R + for , , , Any one of the following; m is 8~20; y is 0~9.

[0007] A method for preparing the above-mentioned modified hyaluronic acid antibacterial material includes the following steps: S1: Add cation exchange resin to an aqueous solution of sodium hyaluronate to perform ion exchange, converting sodium hyaluronate into protonated hyaluronic acid. Stir, filter to remove cation exchange resin, and freeze-dry to obtain acidified hyaluronic acid (HA-AA). S2: Prepare an aqueous solution of HA-AA, add quaternary ammonium hydroxide, stir to carry out acid-base neutralization reaction, and utilize the carboxylate anion of HA (-COO) - ) and quaternary ammonium cations (-R4N) of quaternary ammonium salts (QAS) + The electrostatic interaction between the two is used to obtain a hyaluronic acid-quaternary ammonium salt (HA-QAS) electrostatic complex; freeze drying yields a hyaluronic acid electrostatically grafted quaternary ammonium salt antibacterial material.

[0008] Furthermore, the molecular weight of the sodium hyaluronate described in S1 is 5~3000 kDa; preferably 5~2190 kDa.

[0009] Furthermore, the cation exchange resin mentioned in S1 is any one or a combination of two of the following: 732 hydrogen-type strong acid cation exchange resin (Shanghai Aladdin Biochemical Technology Co., Ltd.) and Amberlite IRN-77 strong acid cation exchange resin (Thermo Fisher Scientific).

[0010] Furthermore, the mass ratio of sodium hyaluronate to cation exchange resin in S1 is 1:5 to 1:15.

[0011] Furthermore, the stirring time in S1 is 6~24 h; the freeze-drying time is 24~72 h, and the temperature is -80℃~50℃.

[0012] Furthermore, the concentration of the aqueous solution of HA-AA in S2 is 0.005~0.05 g / mL.

[0013] Further, the quaternary ammonium hydroxide mentioned in S2 is one or a combination of at least two of tetramethylammonium hydroxide (TMAOH), tetraethylammonium hydroxide (TEAOH), tetrapropylammonium hydroxide (TPrAOH), tetrabutylammonium hydroxide (TBAOH), tetrapentylammonium hydroxide (TPeAOH), tetrahexylammonium hydroxide (THAOH), benzalkonium chloride hydroxide (BACOH), benzyl chloride hydroxide (BZTOH), and monopyridine quaternary ammonium hydroxide (MPQASOH).

[0014] Furthermore, the molar ratio of HA-AA to quaternary ammonium hydroxide in S2 is 1:1 to 20:1.

[0015] Furthermore, the acid-base neutralization reaction described in S2 takes 6 to 24 hours and is carried out at a temperature of 25°C.

[0016] Furthermore, the freeze-drying time described in S2 is 24~72 h, and the temperature is -80℃~-50℃.

[0017] Furthermore, in the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material described in S2, the molar substitution degree of the quaternary ammonium group is 1%~100%.

[0018] An antibacterial hydrogel is prepared from the above-mentioned modified hyaluronic acid antibacterial material.

[0019] A method for preparing the above-mentioned antibacterial hydrogel includes the following preparation steps: A cross-linking agent and glacial acetic acid were added to an aqueous solution of an electrostatically grafted quaternary ammonium salt antibacterial material to carry out a cross-linking reaction. After dialysis, an antibacterial hydrogel was obtained.

[0020] Further, the crosslinking agent is any one or at least a combination of two of 1,4-butanediol diglycidyl ether (BDDE), four-arm polyethylene glycol propylene oxide (4-arm PEG-Ep), and eight-arm polyethylene glycol propylene oxide (8-arm PEG-Ep); preferably BDDE.

[0021] Furthermore, the molar ratio of the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material, the crosslinking agent, and glacial acetic acid is 1:1~20:1~20; preferably 1:5~1:10.

[0022] Furthermore, the cross-linking reaction takes 4-6 hours and is carried out at a temperature of 40-60°C.

[0023] Furthermore, the dialysis medium is phosphate-buffered saline (PBS) with pH=7.4, the dialysis membrane has a molecular weight cutoff of 500 Da~10 kDa, the dialysis temperature is 25℃, and the dialysis time is 24~72 h.

[0024] An application of the above-mentioned antibacterial hydrogel is in the field of hydrogel dressings.

[0025] The hydrogel dressing is an anti-infective wound dressing, an acne care dressing, a skin repair dressing, and a biomedical antibacterial dressing.

[0026] Compared with existing technologies, this invention solves the technical problems of existing hyaluronic acid-based antibacterial materials having weak antibacterial activity, narrow antibacterial spectrum, and easy dependence on oxidative modification leading to damage to the molecular structure and bioactivity of hyaluronic acid, as well as existing nano-silver antibacterial dressings having biotoxicity and poor cell compatibility. Existing technologies cannot simultaneously achieve broad-spectrum antibacterial performance, thermal stability, biocompatibility, and wound repair capabilities. The specific beneficial effects are as follows: 1. This invention constructs modified hyaluronic acid antibacterial materials by controlling the alkyl chain length and degree of quaternization of quaternary ammonium salts, achieving precise regulation of antibacterial activity. These materials exhibit highly efficient and broad-spectrum antibacterial effects against different strains (Gram-negative bacteria, Gram-positive bacteria, and Propionibacterium acnes). Specifically, the antibacterial activity of medium- and long-chain derivatives significantly increases with increasing degree of substitution, with the MIC value decreasing to trace levels. Specific derivatives such as HA-BAC, HA-BZT, and HA-MPQAS maintain excellent antibacterial properties even at low degrees of substitution, combining broad-spectrum antibacterial potential with the advantage of high-efficiency antibacterial activity at low doses. This effectively avoids the inactivation problem of short-chain derivatives at high degrees of substitution, achieving a balance between antibacterial activity, material stability, and biosafety, and significantly improving the balance between activity and safety at low dosages. Simultaneously, it avoids the problem of damaged molecular structure and bioactivity associated with traditional hyaluronic acid modification relying on oxidation. The antibacterial material provided by this invention combines the good biocompatibility of hyaluronic acid with the potent antibacterial properties of quaternary ammonium salts. It has excellent water solubility and can reduce the irritation caused by high concentrations of antibacterial agents while reducing bacterial load. It provides a new technical solution for developing efficient, safe, and biodegradable antibacterial materials for acne and has good application prospects in the field of medical dressings.

[0027] 2. This invention utilizes modified hyaluronic acid antibacterial materials to prepare an antibacterial hydrogel possessing high antibacterial activity, excellent biocompatibility, and low cytotoxicity. The antibacterial hydrogel exhibits antibacterial efficiencies exceeding 90% against *Escherichia coli*, *Staphylococcus aureus*, and *Propionibacterium acnes*, demonstrating excellent broad-spectrum antibacterial activity against Gram-negative bacteria, Gram-positive bacteria, and acne-related pathogens, effectively inhibiting bacterial proliferation and reducing the risk of infection. The hyaluronic acid matrix endows the material with good hydrophilicity, biocompatibility, and cell-friendly properties, while the long-chain quaternary ammonium salt structure provides stable and efficient antibacterial capabilities. The synergistic effect of these two components achieves a balance between antibacterial performance and cellular safety. Compared to traditional high-dose antibacterial systems, this invention reduces potential irritation and cytotoxicity while maintaining antibacterial efficacy. Therefore, the antibacterial hydrogel provided by this invention has promising application prospects in anti-infective wound dressings, acne care dressings, skin repair dressings, and other biomedical antibacterial dressings.

[0028] 3. This invention achieves grafting of hyaluronic acid and quaternary ammonium salt through electrostatic interaction, which can endow the material with excellent antibacterial properties while maintaining the structural integrity of the hyaluronic acid polysaccharide. The antibacterial material and antibacterial hydrogel provided by this invention have mild reaction conditions, simple processes, and convenient purification. Moreover, the obtained material has good water solubility and stability, which can effectively avoid the destruction of the hyaluronic acid structure and biological activity caused by traditional chemical modification, and is suitable for large-scale preparation and application. Attached Figure Description

[0029] Figure 1 The images show the colony growth of the hyaluronic acid electrostatically grafted quaternary ammonium salt antibacterial materials prepared in Examples 7-12 after antibacterial treatment with Propionibacterium acnes. Figure 2 This is a schematic diagram of the reaction structure of the antibacterial hydrogel; Figure 3 This is a diagram showing the colony growth of the antibacterial hydrogel prepared in Example 16 after it has been treated with Escherichia coli, Staphylococcus aureus and Propionibacterium acnes. Figure 4 The cell survival rate of mouse fibroblasts after treatment with antibacterial hydrogel extract at different volume percentages; Figure 5 Fluorescence microscopy image of a live / dead cell staining experiment based on antibacterial hydrogel. Detailed Implementation

[0030] To make the technical solutions of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. It should be noted that the following embodiments are only used to better understand the technical solutions of the present invention and should not be construed as limiting the present invention.

[0031] Example 1. Add 10 g of sodium hyaluronate (M w =259 kDa) was dissolved in 2L of deionized water, and 100 g of 732 hydrogen-type strong acid cation exchange resin (Shanghai Aladdin Biochemical Technology Co., Ltd.) was added. The mixture was stirred at room temperature for 24 h, filtered to remove the cation exchange resin, and freeze-dried at -50℃ for 48 h to obtain HA-AA.

[0032] Example 2. The HA-AA (0.6 g, 1.6 mmol) prepared in Example 1 was dissolved in 60 mL of deionized water. TMAOH (0.23 g, 0.64 mmol) was added under stirring and room temperature conditions to carry out an acid-base neutralization reaction for 24 h to obtain a hyaluronic acid-quaternary ammonium salt (HA-QAS) electrostatic complex. The complex was then freeze-dried at -50 °C for 48 h to obtain the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TMA-1 (259 kDa, degree of substitution 40%).

[0033] Example 3. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide was changed to TEAOH, and the amount was 0.47 g (0.8 mmol). The rest of the preparation steps and conditions were the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TEA-1 (259 kDa, degree of substitution 46%) was obtained.

[0034] Example 4. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide is adjusted to TPrAOH, and the amount is 0.41 g (0.8 mmol). The remaining preparation steps and conditions are the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TPrA-1 (259 kDa, degree of substitution 46%) is obtained.

[0035] Example 5. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide is adjusted to TBAOH, and the amount is 0.41 g (0.8 mmol). The remaining preparation steps and conditions are the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TBA-1 (259 kDa, degree of substitution of 42%) is obtained.

[0036] Example 6. The difference between this embodiment and Example 5 is that the amount of TBAOH was adjusted to 0.75 g (1.44 mmol), while the rest of the preparation steps and conditions were the same as in Example 5, resulting in hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TBA-2 (259 kDa, degree of substitution 80%).

[0037] Example 7. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide is adjusted to TPeAOH, and the amount is 1.26 g (0.8 mmol). The remaining preparation steps and conditions are the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TPeA-1 (259 kDa, degree of substitution of 39%) is obtained.

[0038] Example 8. The difference between this embodiment and Example 7 is that the amount of TPeAOH was adjusted to 2.27 g (1.44 mmol), while the rest of the preparation steps and conditions were the same as in Example 7, resulting in the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TPeA-2 (259 kDa, degree of substitution 90%).

[0039] Example 9. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide is adjusted to THAOH, and the amount is 0.03 g (0.08 mmol). The remaining preparation steps and conditions are the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-THA-1 (259 kDa, degree of substitution of 5%) is obtained.

[0040] The preparation steps of THAOH are as follows: 711 gel-type strong basic anion exchange resin is immersed in a saturated sodium chloride solution for 24 hours. The saturated sodium chloride solution is then discarded, and the resin is repeatedly rinsed with deionized water until the elution is colorless. Six times the volume of 711 gel-type strong basic anion exchange resin (2% by mass) of dilute hydrochloric acid is added, and the resin is immersed for 6 hours. After filtering out the acid, the resin is continuously rinsed with deionized water until the pH of the elution solution is neutral. Then, six times the volume of resin (2% by mass) of sodium hydroxide solution is added, and the resin is immersed for 6 hours. After filtering out the alkaline solution, the resin is further rinsed with deionized water until the pH of the elution solution is neutral, yielding the pretreated ion exchange resin, which is then sealed for later use.

[0041] 10 g of THABr was dissolved in 100 mL of a methanol and water mixture (volume ratio 1:1), and 100 g of pretreated ion exchange resin was added for ion exchange. The resin column was then washed with a methanol / water mixture (volume ratio 1:1) until the pH of the wash solution was neutral. The methanol solvent was removed by rotary evaporation, and the solution was freeze-dried to obtain THAOH.

[0042] Example 10. The difference between this embodiment and Example 9 is that the amount of THAOH was adjusted to 0.06 g (0.16 mmol), while the rest of the preparation steps and conditions were the same as in Example 9, resulting in hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-THA-2 (259 kDa, degree of substitution 11%).

[0043] Example 11. The difference between this embodiment and Example 9 is that the amount of THAOH was adjusted to 0.15 g (0.4 mmol), while the other preparation steps and conditions were the same as in Example 9, resulting in hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-THA-3 (259 kDa, degree of substitution 23%).

[0044] Example 12. The difference between this embodiment and Example 9 is that the amount of THAOH was adjusted to 0.27 g (0.72 mmol), while the rest of the preparation steps and conditions were the same as in Example 9, resulting in hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-THA-4 (259 kDa, degree of substitution 44%).

[0045] Example 13. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide is adjusted to BACOH, and the amount is 0.027 g (0.08 mmol). The remaining preparation steps and conditions are the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-BAC (259 kDa, degree of substitution of 5%) is obtained.

[0046] The difference between the preparation steps of BACCOH and those of THAOH in Example 9 is that BACCl is used instead of THABr, while the other preparation steps remain unchanged.

[0047] Example 14. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide is adjusted to BZTOH, and the amount is 0.04 g (0.08 mmol). The remaining preparation steps and conditions are the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-BZT (259 kDa, degree of substitution of 5%) is obtained.

[0048] The difference between the preparation steps of BZTOH and the preparation steps of THAOH in Example 9 is that BZTCl is used instead of THABr, while the other preparation steps remain unchanged.

[0049] Example 15. The difference between this embodiment and Example 2 is that the quaternary ammonium hydroxide is adjusted to MPQASOH, and the amount is 0.027 g (0.08 mmol). The remaining preparation steps and conditions are the same as in Example 2, and the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-MPQAS (259 kDa, degree of substitution of 5%) is obtained.

[0050] The difference between the preparation steps of MPQASOH and those of THAOH in Example 9 is that MPQASCl is used instead of THABr, while the other preparation steps remain unchanged.

[0051] Example 16. 40 mg of HA-THA-4 prepared in Example 12 was dissolved in 800 μL of deionized water, and then a homogeneous mixture of 30 μL BDDE and 15 μL glacial acetic acid was added. The mixture was reacted at 40 °C for 4 h. After the reaction, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 5 kDa and dialyzed in 1 L, 20 mM PBS buffer (pH 7.4) for 48 h, with the dialysate changed every 4 h. After dialysis, HA-THA antibacterial hydrogel was obtained. The specific synthetic route is as follows: Figure 2 As shown in the figure, x is 44 and n is 683.

[0052] Comparative Example 1. The difference between this comparative example and Example 2 is that the amount of TMAOH was adjusted to 0.47 g (1.28 mmol), while the rest of the preparation steps and conditions were the same as in Example 2, resulting in hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TMA-2 (259 kDa, degree of substitution 75%).

[0053] Comparative Example 2. The difference between this comparative example and Example 3 is that the amount of TEAOH was adjusted to 0.85 g (1.44 mmol), while the rest of the preparation steps and conditions were the same as in Example 3, resulting in hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TEA-2 (259 kDa, degree of substitution 85%).

[0054] Comparative Example 3. The difference between this embodiment and Example 4 is that the amount of TPrAOH was adjusted to 0.73 g (1.44 mmol), while the rest of the preparation steps and conditions were the same as in Example 4, resulting in the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material HA-TPrA-2 (259 kDa, degree of substitution 82%).

[0055] (I) In vitro antibacterial activity test of hyaluronic acid electrostatically grafted quaternary ammonium salt antibacterial material: This invention systematically evaluated the in vitro antibacterial activity of hyaluronic acid electrostatically grafted quaternary ammonium salt (HA-QAS) antibacterial materials with different alkyl chain lengths and degrees of substitution (DS). The minimum inhibitory concentrations (MICs) against representative Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) were tested using the broth microdilution method. The test results are shown in Table 1. For the short-chain derivatives HA-TMA, HA-TEA, and HA-TPrA prepared in Examples 2-4, when the DS was 40%–46%, they all showed an MIC of 16 mg / mL against Escherichia coli and an MIC of 8 mg / mL against Staphylococcus aureus, demonstrating certain antibacterial activity. However, when the dissociation degree (DS) of the short-chain derivatives HA-TMA, HA-TEA, and HA-TPrA prepared in Comparative Examples 1-3 increased to 75%–85%, the antibacterial effect decreased significantly, with MICs all exceeding 16 mg / mL, indicating near-complete loss of activity. This suggests that excessively high substitution degrees in short-chain derivatives may lead to molecular aggregation or reduced solubility, thereby inhibiting antibacterial activity.

[0056] The medium-chain alkyl derivatives HA-TBA and HA-TPeA provided in Examples 5-8 exhibited a significant substitution-dependent enhancement effect. HA-TBA showed an increasing MIC against *Escherichia coli* as the DS increased from 42% to 80%, while the MIC against *Staphylococcus aureus* decreased significantly from 16 mg / mL to 2 mg / mL, indicating that the antibacterial activity of QAS is more sensitive to positive bacteria. HA-TPeA, at DS of 39% and 90%, had MICs of 8 mg / mL and 2 mg / mL against *Escherichia coli*, respectively, and 0.25 mg / mL and 0.125 mg / mL against *Staphylococcus aureus*, respectively, demonstrating that medium- and long-chain derivatives can achieve potent antibacterial activity at low concentrations and have significant effects against both types of bacteria.

[0057] The long-chain hydrophobic derivative HA-THA prepared in Examples 9-12 was tested using DS gradients of 5%, 11%, 23%, and 44%. The results showed that the MIC decreased significantly with increasing DS. The MIC against *Escherichia coli* decreased from 8 mg / mL to 0.25 mg / mL, and the MIC against *Staphylococcus aureus* decreased from 0.032 mg / mL to 0.004 mg / mL, demonstrating extremely strong antibacterial activity. This proves that the binding of long-chain hydrophobic groups to quaternary ammonium salts can significantly enhance the interaction with bacterial cell membranes, thereby improving antibacterial efficiency and achieving the goal of inhibiting bacteria with minimal dosage.

[0058] Furthermore, the HA-BAC, HA-BZT, and HA-MPQAS prepared in Examples 13-15 exhibited excellent antibacterial activity at low substitution levels (5%), with MICs of 0.031 mg / mL, 0.067 mg / mL, and 0.125 mg / mL against *Escherichia coli*, and 0.001 mg / mL, 0.005 mg / mL, and 0.008 mg / mL against *Staphylococcus aureus*, respectively. These data fully demonstrate that, through specific structural design, highly efficient inhibition of Gram-negative and Gram-positive bacteria can be achieved at extremely low concentrations, providing a potential low-toxicity and highly effective antibacterial solution for practical applications.

[0059] Therefore, by rationally designing the alkyl chain length and degree of substitution, this invention successfully achieves highly efficient antibacterial effects against different types of bacterial strains, realizing broad-spectrum antibacterial activity against both Gram-negative and Gram-positive bacteria. Specifically, the antibacterial activity of medium- and long-chain derivatives is significantly enhanced with increasing substitution degree, and the MIC value is reduced to trace levels. Specific derivatives (HA-BAC, HA-BZT, HA-MPQAS) maintain highly efficient antibacterial activity even at low substitution degrees. This effectively avoids the problem of short-chain derivatives becoming inactive at high substitution degrees, achieving an optimized balance between antibacterial activity and material stability, and significantly improving the balance between the activity and safety of antibacterial materials at low dosages.

[0060] Table 1

[0061] (II) Antibacterial activity test of hyaluronic acid electrostatically grafted quaternary ammonium salt antibacterial material against Propionibacterium acnes: To further verify the inhibitory effect of the hyaluronic acid electrostatic grafted quaternary ammonium salt (HA-QAS) antibacterial material of this invention on acne-related pathogens, *Propionibacterium acnes* (P. acnes) was used as a model strain. The antibacterial properties of the HA-QAS antibacterial materials prepared in Examples 7-12 were systematically evaluated using the plate count method and the broth microdilution method. *Propionibacterium acnes* was inoculated into agar plates, and the antibacterial effect of the HA-QAS antibacterial material was investigated at a concentration of 16 mg / mL. Figure 1 The image shows the colony growth of *Propionibacterium acnes* after treatment with HA-QAS antibacterial material. The bacterial solution without HA-QAS antibacterial material was used as a control group. The agar plate surface of the control group was covered with a large number of colonies, indicating that *Propionibacterium acnes* could grow and reproduce normally. However, after treatment with HA-QAS antibacterial material, the number of colonies on the agar plate was significantly reduced, and some samples showed almost no visible colony formation. This demonstrates that the HA-QAS antibacterial material provided by this invention can effectively inhibit the proliferation of *Propionibacterium acnes*, directly verifying that HA-QAS antibacterial material has excellent bactericidal or bacteriostatic ability against acne-related pathogens.

[0062] The minimum inhibitory concentrations (MICs) of the HA-TPeA and HA-THA antibacterial materials in Examples 7-12 against *Propionibacterium acnes* were further determined using the broth microdilution method, and the results are shown in Table 2. The antibacterial activity of the HA-QAS material against *Propionibacterium acnes* significantly increased with increasing quaternary ammonium salt alkyl chain length and degree of quaternization. Specifically, the MICs of the HA-TPeA antibacterial material against *Propionibacterium acnes* were 2 mg / mL and 0.5 mg / mL at quaternization degrees of 39% and 90%, respectively, indicating that increasing the degree of quaternization significantly enhanced its antibacterial performance. In the HA-THA antibacterial material, which has a longer hydrophobic chain structure, as the degree of quaternization gradually increased from 5% to 44%, its MIC against *Propionibacterium acnes* decreased sequentially from 0.256 mg / mL to 0.128 mg / mL, 0.064 mg / mL, and 0.032 mg / mL, showing a clear concentration-dependent increasing trend. In particular, HA-THA-4 can achieve highly efficient inhibition of Propionibacterium acnes even at low concentrations, indicating a significant synergistic effect between long-chain hydrophobic groups and quaternary ammonium salt structures, which can effectively enhance the interaction between the material and the bacterial cell membrane, thereby improving antibacterial efficiency.

[0063] This invention demonstrates that the HA-QAS antibacterial material provided by this invention not only effectively inhibits the growth of Propionibacterium acnes, but also achieves precise control of antibacterial activity by regulating the alkyl chain length and degree of quaternization of the quaternary ammonium salt, exhibiting excellent broad-spectrum antibacterial potential and the advantages of high-efficiency, low-dose antibacterial action. Furthermore, the antibacterial material provided by this invention combines the good biocompatibility of hyaluronic acid with the potent antibacterial properties of quaternary ammonium salts, reducing bacterial load while minimizing irritation caused by the use of high-concentration antibacterial agents. This provides a new technical solution for developing highly efficient, safe, and biodegradable antibacterial materials specifically for acne, and shows promising application prospects in the field of medical dressings.

[0064] Table 2

[0065] (III) Antibacterial activity test of HA-THA antibacterial hydrogel: The HA-THA antibacterial hydrogel prepared in Example 16 was used to test its antibacterial activity against Escherichia coli, Staphylococcus aureus, and Propionibacterium acnes. Bacterial solutions without HA-THA antibacterial hydrogel were used as control groups. The test results are as follows: Figure 3As shown in the figure, a large number of clearly visible colonies grew on the surface of the agar culture plates in the control group, indicating that all three strains could grow and reproduce normally. After treatment with HA-THA antibacterial hydrogel, the number of colonies in the culture plates of each experimental group was significantly reduced. The antibacterial efficiency of HA-THA antibacterial hydrogel against Escherichia coli, Staphylococcus aureus and Propionibacterium acnes reached 90%, 99% and 99% respectively, indicating that the HA-THA antibacterial hydrogel provided by the present invention can effectively inhibit bacterial proliferation and has excellent broad-spectrum antibacterial activity against Gram-positive bacteria, Gram-negative bacteria and acne-related pathogens, which can effectively reduce the risk of bacterial infection.

[0066] (iv) Cytotoxicity test of HA-THA antibacterial hydrogel: To assess the biocompatibility of the HA-THA antibacterial hydrogel provided by this invention, in vitro cytotoxicity tests were conducted on mouse fibroblasts (L929) using the MTT assay. The HA-THA antibacterial hydrogel prepared in Example 16 was placed in sterile cell culture medium and extracted at 37°C for 24 h to obtain a hydrogel extract. Subsequently, hydrogel extracts with volume percentages of 10%, 25%, 50%, 75%, and 100% were prepared. Mouse fibroblasts (L929) in the logarithmic growth phase were seeded into 96-well plates and cultured at 37°C in a 5% CO2 incubator for 24 h to stabilize cell adhesion. The culture medium was then replaced with different concentrations of hydrogel extract and cultured for another 24 h. After culture, MTT solution was added to each well, and incubation was continued for 4 h. The supernatant was discarded, and DMSO was added to dissolve the crystallized precipitate. The absorbance at 492 nm was measured using a microplate reader. Cell viability was calculated based on the absorbance of the experimental group and the control group to evaluate the cytotoxicity of the HA-THA antibacterial hydrogel.

[0067] like Figure 4 The figure shows the cell viability of L929 cells after treatment with different volume percentages of HA-THA-4 antibacterial hydrogel leachate. As can be seen from the figure, the cell viability gradually decreases with increasing hydrogel leachate concentration. Specifically, at 10% and 25% volume percentages, the cell viability is above 90% and 80%, respectively, indicating that low concentrations of HA-THA-4 antibacterial hydrogel leachate have little impact on cell growth and exhibit good cell compatibility. When the leachate concentration is further increased to 50%, 75%, and 100%, although the cell viability decreases somewhat, it still remains at a high level (above 60%), proving that the HA-THA-4 antibacterial hydrogel provided by this invention does not exhibit significant strong cytotoxicity.

[0068] The fluorescence of L929 cells was observed using the Calcein-AM / propidium iodide (PI) live / dead cell staining method. Figure 5The image shows a fluorescence microscope image of a live / dead cell staining experiment based on HA-THA-4 antibacterial hydrogel. As can be seen from the image, the cell morphology and density of the HA-THA-4 antibacterial hydrogel-treated group are similar to those of the control group. Almost all cells express green fluorescence, with only a small amount of red fluorescence, demonstrating that the HA-THA-4 antibacterial hydrogel has no significant cytotoxicity and good cell compatibility. This result is consistent with the MTT assay results, indicating that the HA-THA antibacterial hydrogel constructed based on hyaluronic acid electrostatic grafted quaternary ammonium salt possesses both excellent antibacterial properties and good biocompatibility. The hyaluronic acid matrix endows the material with good hydrophilicity, biocompatibility, and cell-friendly properties, while the long-chain quaternary ammonium salt structure provides highly efficient and stable antibacterial capabilities. The two work synergistically to achieve an effective balance between antibacterial performance and cell safety. Therefore, the antibacterial hydrogel provided by this invention has promising application prospects in the fields of anti-infective wound dressings, acne care dressings, skin repair dressings, and other biomedical antibacterial dressings.

[0069] In summary, this invention constructs a modified hyaluronic acid antibacterial material by controlling the alkyl chain length and degree of quaternization of the quaternary ammonium salt, achieving precise regulation of antibacterial activity. It exhibits highly efficient and broad-spectrum antibacterial effects against different strains (Gram-negative bacteria, Gram-positive bacteria, and Propionibacterium acnes). Simultaneously, an antibacterial hydrogel with high antibacterial activity, excellent biocompatibility, and low cytotoxicity is prepared using the modified hyaluronic acid antibacterial material. The hyaluronic acid matrix endows the material with good hydrophilicity, biocompatibility, and cell-friendly properties, while the long-chain quaternary ammonium salt structure provides stable and efficient antibacterial capabilities. The two work synergistically to achieve a balance between antibacterial performance and cellular safety. The antibacterial material and antibacterial hydrogel preparation method provided by this invention have mild reaction conditions, simple processes, and convenient purification. Furthermore, the obtained material exhibits good water solubility and stability, effectively avoiding the destruction of the hyaluronic acid structure and bioactivity caused by traditional chemical modification, making it suitable for large-scale preparation and application.

[0070] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0071] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A modified hyaluronic acid antibacterial material, characterized in that, The structural formula is shown in Formula I: Formula I; In the formula, n is 10~6000; x / n×100%=1%~99%; R + for , , , Any one of the following; m is 8~20; y is 0~9.

2. A method for preparing the modified hyaluronic acid antibacterial material as described in claim 1, characterized in that, Includes the following steps: S1: Add cation exchange resin to an aqueous solution of sodium hyaluronate, stir, filter, and freeze-dry to obtain acidified hyaluronic acid; S2: Prepare an aqueous solution of acidified hyaluronic acid, add quaternary ammonium hydroxide, stir to carry out acid-base neutralization reaction, and obtain hyaluronic acid-quaternary ammonium salt electrostatic complex; freeze dry to obtain hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material.

3. The preparation method of the modified hyaluronic acid antibacterial material according to claim 2, characterized in that, The sodium hyaluronate in S1 has a molecular weight of 5~3000 kDa; the cation exchange resin is any one or a combination of two of 732 hydrogen-type strong acid cation exchange resin and Amberlite IRN-77 strong acid cation exchange resin; the mass ratio of sodium hyaluronate to cation exchange resin is 1:5~1:15; the stirring time is 6~24 h; the freeze-drying time is 24~72 h, and the temperature is -80℃~-50℃.

4. The preparation method of the modified hyaluronic acid antibacterial material according to claim 2, characterized in that, The concentration of the aqueous solution of acidified hyaluronic acid in S2 is 0.005~0.05 g / mL; the quaternary ammonium hydroxide is one or a combination of at least two of tetramethylammonium hydroxide, tetraethylammonium hydroxide, tetrapropylammonium hydroxide, tetrabutylammonium hydroxide, tetrapentylammonium hydroxide, tetrahexylammonium hydroxide, benzalkonium chloride hydroxide, benzyl chloride hydroxide, and monopyridine quaternary ammonium hydroxide; the molar ratio of acidified hyaluronic acid to quaternary ammonium hydroxide is 1:1~20:

1.

5. The preparation method of the modified hyaluronic acid antibacterial material according to claim 2, characterized in that, The acid-base neutralization reaction in S2 takes 6-24 hours at a temperature of 25°C; the freeze-drying takes 24-72 hours at a temperature of -80°C to -50°C; and the molar substitution degree of the quaternary ammonium group in the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material is 1%-100%.

6. An antibacterial hydrogel, characterized in that, It is prepared from the modified hyaluronic acid antibacterial material according to claim 1.

7. A method for preparing the antibacterial hydrogel as described in claim 6, characterized in that, The preparation steps include the following: A cross-linking agent and glacial acetic acid were added to an aqueous solution of an electrostatically grafted quaternary ammonium salt antibacterial material to carry out a cross-linking reaction. After dialysis, an antibacterial hydrogel was obtained.

8. The method for preparing the antibacterial hydrogel according to claim 7, characterized in that, The crosslinking agent is any one or at least a combination of two of 1,4-butanediol diglycidyl ether, tetra-arm polyethylene glycol propylene oxide, and octa-arm polyethylene glycol propylene oxide; the molar ratio of the hyaluronic acid electrostatic grafted quaternary ammonium salt antibacterial material, the crosslinking agent, and glacial acetic acid is 1:1~20:1~20.

9. The method for preparing the antibacterial hydrogel according to claim 7, characterized in that, The cross-linking reaction takes 4-6 hours and is carried out at a temperature of 40-60°C. The dialysis medium is a phosphate buffer solution with a pH of 7.

4. The dialysis membrane has a molecular weight cutoff of 500 Da to 10 kDa, the dialysis temperature is 25°C, and the dialysis time is 24-72 hours.

10. The application of the antibacterial hydrogel as described in claim 6, characterized in that, It is used in the field of hydrogel dressings.

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