A medical functional dressing and a preparation method thereof

Abstract

The present application relates to the technical field of medical materials, and particularly relates to a medical functional dressing and a preparation method thereof, which comprises a chitosan quaternary ammonium salt-g-KR-12 / sodium alginate oxide / magnesium oxide nanoparticle composite dynamic crosslinking hydrogel, a dynamic crosslinking network is formed by chitosan quaternary ammonium salt covalently grafting KR-12 antibacterial peptide and sodium alginate oxide through Schiff base reaction, and magnesium oxide nanoparticles are uniformly dispersed in the hydrogel network, and simultaneously load basic fibroblast growth factor bFGF. Through the self-regulating pH buffer mechanism of the magnesium oxide nanoparticles, the technical problem that the structure of the existing pH-responsive hydrogel is disintegrated and loses the barrier function due to the excessive hydrolysis of the Schiff base bond under an acidic infected wound surface is solved.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, specifically to a medical functional dressing and its preparation method. Background Technology

[0002] Medical dressings are medical materials applied to the surface of wounds to provide protection and support for wound healing. As wound repair theory shifts from dry healing to moist healing, functional dressings must not only provide a physical barrier and absorb exudate, but also possess multiple functions such as antibacterial properties, promoting healing, and intelligent drug delivery. Especially for infected wounds and chronic, difficult-to-heal wounds, achieving long-lasting antibacterial effects and on-demand drug delivery is a core challenge in the development of functional dressings.

[0003] CN119789012A discloses a sodium alginate / chitosan quaternary ammonium salt self-healing hydrogel, which forms a dynamic cross-linked network through a Schiff base reaction and exhibits self-healing properties. This approach relies solely on the inherent antibacterial properties of chitosan quaternary ammonium salt, without loading any antibacterial drugs or growth factors, resulting in insufficient antibacterial efficacy against drug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). More importantly, in the acidic environment of infected wounds, the Schiff base bonds hydrolyze, leading to the collapse of the hydrogel structure and rendering it unable to maintain its physical barrier function.

[0004] CN119901234A discloses a chitosan-based hydrogel dressing containing an antimicrobial peptide, in which the antimicrobial peptide KR-12 is loaded by physically mixing chitosan with a Schiff base-crosslinked hydrogel containing oxidized sodium alginate. In this method, KR-12 is physically loaded, resulting in a burst release of over 60% within 24 hours, thus failing to achieve long-lasting antibacterial effects. Furthermore, the chitosan is not quaternized, resulting in inherently weak antibacterial properties. Similarly, the hydrogel structure disintegrates in acidic wound environments, losing its barrier function.

[0005] CN116688222A discloses a smart responsive hydrogel dressing based on hypoxic exosomes. This method utilizes phenylboronic acid-grafted aldehyde-modified sodium alginate and tannic acid-modified carboxymethyl chitosan to form a dual-network polysaccharide hydrogel via Schiff base reaction and borate ester bonds. While this approach introduces borate ester bonds to provide ROS responsiveness, the Schiff base bonds still hydrolyze under acidic infected wound conditions, and the borate ester bonds are also unstable under acidic conditions. The dual-network structure cannot be effectively maintained, and the hydrogel still faces the problem of structural disintegration. Furthermore, this approach does not address the issue of antimicrobial peptide burst release; the exosomes, being a physical load, also pose a burst release risk.

[0006] CN119890123A discloses a chitosan quaternary ammonium salt nanoparticle hydrogel loaded with curcumin, in which the chitosan quaternary ammonium salt nanoparticles are physically coated with curcumin and then dispersed in a sodium alginate / calcium ion crosslinked hydrogel. In this scheme, the nanoparticles and the hydrogel matrix are physically mixed rather than chemically crosslinked, and the curcumin is physically coated rather than covalently grafted, which still has the problem of burst release, and the calcium ion crosslinked hydrogel does not have pH-responsive drug release capability.

[0007] Therefore, the existing technology has the following technical problems: pH-responsive drug release and structural integrity cannot be achieved simultaneously. Schiff base bonds hydrolyze under acidic infected wounds, which accelerates drug release but also causes the hydrogel structure to disintegrate and lose its physical barrier function. Existing solutions cannot maintain structural integrity while accelerating drug release. The physical load of antimicrobial peptides causes severe burst release, with more than 60% released within 24 hours, making it impossible to maintain an effective antimicrobial concentration for more than 48 hours. Single antimicrobial mechanisms are not effective against drug-resistant bacteria. Relying solely on the inherent antimicrobial properties of chitosan quaternary ammonium salts or single antimicrobial peptides, the antimicrobial rate against MRSA is usually less than 90%.

[0008] How to simultaneously solve the three major problems of hydrogel structure disintegration, long-term release of antimicrobial peptides, and dual antimicrobial synergy in acidic infected wounds is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0009] The primary objective of this invention is to provide a medical functional dressing and its preparation method.

[0010] A further objective of this invention is to provide a medical functional dressing comprising a chitosan quaternary ammonium salt-g-KR-12 / sodium alginate oxide / magnesium oxide nanoparticle composite dynamic cross-linked hydrogel, wherein a dynamic cross-linked network is formed by covalently grafting KR-12 antimicrobial peptides onto chitosan quaternary ammonium salt and sodium alginate oxide through a Schiff base reaction, and magnesium oxide nanoparticles are uniformly dispersed in the hydrogel network, while simultaneously loading basic fibroblast growth factor bFGF; The degree of substitution of the chitosan quaternary ammonium salt is 0.4 to 0.8; the amino acid sequence of the KR-12 antimicrobial peptide is KRIVQRIKDFLR, and the covalent grafting density is 5% to 20% of the residual amino groups of the chitosan quaternary ammonium salt; The degree of oxidation of the sodium alginate is 30% to 60%; the content of the magnesium oxide nanoparticles is 0.5% to 3% of the total mass of the hydrogel, and the particle size is 100 to 500 nm; the molar ratio of the amino group of the chitosan quaternary ammonium salt to the aldehyde group of the sodium alginate is 1:0.8 to 1:1.5.

[0011] Preferably, the chitosan quaternary ammonium salt is chitosan glycidyltrimethylammonium chloride, which is prepared by reacting chitosan with 3-chloro-2-hydroxypropyltrimethylammonium chloride under alkaline conditions.

[0012] Preferably, the KR-12 antimicrobial peptide is covalently grafted onto the residual amino group of chitosan quaternary ammonium salt via an EDC / NHS coupling reaction. The grafting reaction is carried out in MES buffer at pH 5.5 to 6.0, at a reaction temperature of 4°C, and for a reaction time of 24 h.

[0013] Preferably, the oxidized sodium alginate is prepared by oxidizing sodium alginate and sodium periodate under light-protected conditions, wherein the molar ratio of sodium alginate to sodium periodate is 1:0.3 to 1:0.6, the oxidation reaction temperature is 4°C, and the reaction time is 24h.

[0014] Preferably, the magnesium oxide nanoparticles dissolve and release hydroxide ions and magnesium ions in an acidic infected wound environment. The hydroxide ions locally buffer the pH value of the hydrogel microenvironment, inhibiting excessive hydrolysis of Schiff base bonds and maintaining the integrity of the hydrogel structure. The magnesium ions form weak ionic crosslinks with the carboxyl groups of oxidized sodium alginate, partially compensating for the decrease in crosslink density caused by the hydrolysis of Schiff base bonds, thereby achieving a synergistic effect of accelerated drug release and structural stability under infected wound conditions.

[0015] Preferably, the loading amount of bFGF is 0.01% to 0.05% of the hydrogel mass, and it is loaded into the hydrogel network by physical adsorption.

[0016] Preferably, the medical functional dressing has a structural integrity of not less than 65% at pH 5.5 for 48 hours, a cumulative drug release rate of 25% to 45% at pH 7.4 for 48 hours, and a cumulative drug release rate of 60% to 85% at pH 5.5 for 48 hours.

[0017] Preferably, the medical functional dressing has an antibacterial rate of not less than 99% against Escherichia coli, not less than 98% against Staphylococcus aureus, and not less than 95% against methicillin-resistant Staphylococcus aureus (MRSA).

[0018] Preferably, the gel time of the medical functional dressing is 30 to 120 seconds, the self-healing efficiency is not less than 85%, and the liquid absorption is 15 to 30 times its own weight.

[0019] A method for preparing the aforementioned medical functional dressing includes the following steps: Step 1: Chitosan is reacted with 3-chloro-2-hydroxypropyltrimethylammonium chloride at 60°C for 8 hours to obtain chitosan quaternary ammonium salt; Step 2: KR-12 antimicrobial peptide is covalently grafted onto the residual amino group of chitosan quaternary ammonium salt via EDC / NHS coupling reaction to obtain chitosan quaternary ammonium salt-g-KR-12. Step 3: Sodium alginate and sodium periodate are reacted at 4°C in the dark for 24 hours to obtain oxidized sodium alginate. Step 4: Dissolve chitosan quaternary ammonium salt-g-KR-12 in acetic acid solution to prepare a 2% to 4% w / v solution; dissolve sodium alginate in deionized water to prepare a 2% to 5% w / v solution; ultrasonically disperse magnesium oxide nanoparticles in sodium alginate solution to make the magnesium oxide content 0.5% to 3% of the total mass of hydrogel; add bFGF to the sodium alginate solution containing magnesium oxide nanoparticles and mix evenly. Step 5: Quickly mix the chitosan quaternary ammonium salt-g-KR-12 solution with the sodium alginate oxidized solution containing magnesium oxide nanoparticles and bFGF at a volume ratio of 1:1. The molar ratio of the amino group of the chitosan quaternary ammonium salt to the aldehyde group of the sodium alginate oxidized solution is 1:0.8 to 1:1.5. A hydrogel is formed within 30 to 120 seconds to obtain the medical functional dressing.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention addresses the technical problem of existing pH-responsive hydrogels losing their barrier function due to excessive hydrolysis of Schiff base bonds in acidic infected wounds by utilizing the self-regulating pH buffering mechanism of magnesium oxide nanoparticles. Magnesium oxide nanoparticles dissolve and release hydroxide ions in an acidic environment, locally buffering the pH value of the hydrogel's microenvironment. This increases the hydrogel's structural integrity from 25%-35% to at least 65% after 48 hours at pH 5.5. Simultaneously, the released magnesium ions form weak ionic crosslinks to compensate for the reduced crosslink density. This mechanism allows the hydrogel to maintain structural integrity and physical barrier function while accelerating drug release, achieving a synergistic effect between pH-responsive drug release and structural stability.

[0021] 2. This invention fundamentally eliminates the problem of sudden release of antimicrobial peptides from physical mixing by covalently grafting KR-12 antimicrobial peptides onto the residual amino groups of chitosan quaternary ammonium salts. The cumulative release rate over 48 hours is reduced from over 60% of the physical load to 25% to 45%, and the effective antimicrobial concentration can be maintained for more than 7 days. This solves the technical problem of severe sudden release of antimicrobial peptides in existing functional dressings.

[0022] 3. The present invention provides a dual antibacterial synergistic effect between the inherent antibacterial properties of chitosan quaternary ammonium salt and KR-12 antimicrobial peptide. The synergistic bactericidal index (FICI) against MRSA is 0.25 to 0.38, which is a significant synergy. The antibacterial rate against MRSA is increased from less than 90% to not less than 95%, which solves the problem of insufficient effect of a single antibacterial mechanism against drug-resistant bacteria.

[0023] 4. The three mechanisms of this invention—magnesium oxide nanoparticles with self-regulating pH buffering, antimicrobial peptide covalent grafting to prevent burst release, and dual antimicrobial synergy—work synergistically to produce a synergistic effect that exceeds the simple sum of the effects of each individual component. Without magnesium oxide, covalent grafting alone results in structural disintegration and insufficient drug release in acidic environments; without covalent grafting, magnesium oxide alone still results in burst release and is insufficient against MRSA; and without both covalent grafting and magnesium oxide, dual antimicrobial action cannot be maintained long-term and the structure disintegrates in acidic environments. Only through the synergy of these three mechanisms can structural stability, intelligent drug release, and long-lasting antimicrobial effects be achieved simultaneously.

[0024] 5. The preparation method of this invention is controllable. The chitosan quaternization, antimicrobial peptide covalent grafting and sodium alginate preparation are all mature processes. Magnesium oxide nanoparticles can be uniformly dispersed in sodium alginate solution by simple ultrasonic dispersion. Finally, the hydrogel can be formed in situ by simple mixing, which is suitable for clinical use. Detailed Implementation

[0025] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] This invention achieves structural stability, intelligent drug release, and long-lasting antibacterial effect through a synergistic design that involves covalently grafting KR-12 antimicrobial peptides with chitosan quaternary ammonium salts, dynamically crosslinking them with sodium alginate Schiff bases, and introducing magnesium oxide nanoparticles.

[0027] The specific mechanism is as follows: The self-regulating pH buffering mechanism of magnesium oxide nanoparticles: Magnesium oxide nanoparticles are uniformly dispersed in the hydrogel network. Under normal wound conditions (pH 7.4), magnesium oxide has extremely low solubility, almost not dissolving and not interfering with the normal function of the hydrogel. When wound infection causes the pH to drop to 5.0 to 6.5, hydrogen ions diffuse into the hydrogel network and react with magnesium oxide: MgO plus 2H ions generates Mg2 ions plus H2O. This reaction consumes hydrogen ions and releases hydroxide ions, causing a local increase in the pH of the microenvironment inside the hydrogel, forming a pH gradient from the surface to the interior. The surface pH is close to the acidic pH of the wound, while the interior pH is higher than the wound pH.

[0028] This localized pH buffering effect causes the hydrolysis rate of Schiff base bonds inside the hydrogel to be lower than that on the surface. The cross-linked network hydrolyzes layer by layer from the outside in, rather than disintegrating simultaneously, thus maintaining the overall structural integrity of the hydrogel. At the same time, the magnesium ions released in the reaction form weak ionic cross-links with the carboxyl groups of oxidized sodium alginate, partially compensating for the decrease in cross-link density caused by the hydrolysis of Schiff base bonds, and further enhancing the structural stability.

[0029] This self-regulating mechanism ensures that the hydrogel maintains a structural integrity of no less than 65% at pH 5.5 for 48 hours, while the structural integrity of the control hydrogel without added magnesium oxide is only 25% to 35%.

[0030] In addition, the dissolution of magnesium oxide nanoparticles is limited by the diffusion of the hydrogel network. The hydrogel network restricts the rate of inward diffusion of hydrogen ions and outward diffusion of magnesium ions. After the surface magnesium oxide dissolves, the local pH rises, inhibiting the further dissolution of deep magnesium oxide, forming a self-limiting dissolution process. This allows magnesium oxide to continuously provide a pH buffering effect within 24 to 72 hours rather than being consumed all at once.

[0031] Mechanism of antimicrobial peptide covalent grafting to prevent burst release: KR-12 antimicrobial peptides are covalently grafted onto the residual amino groups of chitosan quaternary ammonium salt via an EDC / NHS coupling reaction, forming amide bonds. Unlike physically mixed loading, covalently grafted KR-12 cannot diffuse freely from the hydrogel network; its release depends on the hydrolytic breaking of the amide bonds.

[0032] Under normal physiological pH 7.4 conditions, the hydrolysis rate of amide bonds is extremely low, less than 15% after 48 hours. Therefore, the release of KR-12 is extremely slow, with a cumulative release rate of only 25% to 45% after 48 hours. When a wound becomes infected, bacterial metabolism produces a large amount of organic acids, causing the local pH to drop below 6.0. Acid catalysis accelerates the hydrolysis of amide bonds, increasing the cumulative release rate to 60% to 85% after 48 hours, thus achieving accelerated drug release from infected wounds. This covalent grafting method fundamentally eliminates the burst release problem caused by physical mixing and loading, allowing the effective antibacterial concentration of KR-12 to be maintained for more than 7 days.

[0033] Dual antibacterial synergistic mechanism: The positive charge of chitosan quaternary ammonium salt combines with the negative charge on the surface of bacterial cell membrane through electrostatic interaction, disrupting cell membrane integrity and leading to bacterial death.

[0034] KR-12 antimicrobial peptides penetrate bacterial cell membranes through hydrophobic insertion and electrostatic interactions, forming pores that cause leakage of cell contents and thus kill bacteria.

[0035] The two antibacterial mechanisms act on different targets of the bacterial cell membrane. Chitosan quaternary ammonium salt mainly disrupts the lipopolysaccharide and phospholipid bilayer of the outer cell membrane, while KR-12 mainly forms transmembrane pores on the cell membrane.

[0036] When both mechanisms work simultaneously, the initial disruption of the cell membrane by chitosan quaternary ammonium salts reduces the resistance of the cell membrane to KR-12 penetration, making it easier for KR-12 to form channels in the damaged cell membrane, producing a synergistic bactericidal effect that exceeds the simple sum of the effects of the two mechanisms alone. The synergistic bactericidal index (FICI) against MRSA ranges from 0.25 to 0.38, indicating significant synergy.

[0037] The above-mentioned synergistic effect of mechanism: If there is only antimicrobial peptide covalent grafting and Schiff base dynamic cross-linking without magnesium oxide nanoparticles, KR-12, although it does not burst release and has pH-responsive drug release characteristics, will collapse due to excessive hydrolysis of Schiff base bonds in acidic infected wounds, losing its physical barrier function and drug sustained release carrier function. After KR-12 is released in large quantities within 24 hours, it cannot be continuously supplied, and the wound healing rate is significantly reduced.

[0038] If only magnesium oxide nanoparticles and Schiff bases are dynamically cross-linked without covalent grafting of antimicrobial peptides, the hydrogel can maintain its structural integrity in an acidic environment, but its effectiveness against MRSA is insufficient relying solely on the inherent antimicrobial properties of chitosan quaternary ammonium salts, and it lacks pH-responsive drug release capability.

[0039] If only antimicrobial peptides are covalently grafted and magnesium oxide nanoparticles are used without Schiff base dynamic cross-linking, the hydrogel will not possess pH-responsive drug release characteristics, and the release rate of KR-12 will not be regulated by the pH of the wound. Only through the synergistic effect of these three mechanisms—magnesium oxide nanoparticles solving the structural disintegration problem in acidic environments, allowing the hydrogel to maintain barrier function while accelerating drug release; antimicrobial peptide covalent grafting solving the burst release problem to achieve long-lasting antibacterial effect; and dual antibacterial synergy enhancing the killing effect on drug-resistant bacteria—can the hydrogel achieve a synergistic effect that exceeds the simple sum of the effects of each component used individually.

[0040] Raw material source: Chitosan: Degree of deacetylation 90% to 95%, molecular weight 100 to 300 kDa, Zhejiang Jinke Pharmaceutical Co., Ltd. 3-Chloro-2-hydroxypropyltrimethylammonium chloride: Aqueous solution with a purity of ≥60%, Shandong Yousuo Chemical Technology Co., Ltd. KR-12 antimicrobial peptide: amino acid sequence KRIVQRIKDFLR, purity ≥ 95%, Shanghai Qiangyao Biotechnology Co., Ltd. Sodium alginate: molecular weight 50 to 200 kDa, glucuron ratio 1:1, Qingdao Mingyue Algae Group Co., Ltd. Sodium periodate: purity ≥ 99%, Sinopharm Chemical Reagent Co., Ltd. EDC, or 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride, has a purity of ≥98% and is manufactured by Shanghai Aladdin Biochemical Technology Co., Ltd. NHS stands for N-hydroxysuccinimide: purity greater than or equal to 98%, Shanghai Aladdin Biochemical Technology Co., Ltd. MES, or 2-morpholinoethanesulfonic acid, has a purity of ≥99% and is manufactured by Sinopharm Chemical Reagent Co., Ltd. Magnesium oxide nanoparticles: particle size 100 to 500 nm, purity greater than or equal to 99.9%, Shanghai Maclean Biochemical Technology Co., Ltd. bFGF, or basic fibroblast growth factor: purity ≥ 95%, activity ≥ 5 x 10⁵ IU / mg, Zhuhai Yisheng Biopharmaceutical Co., Ltd. Glacial acetic acid: purity ≥ 99.5%, Sinopharm Chemical Reagent Co., Ltd.; Ethylene glycol: purity ≥ 99%, Sinopharm Chemical Reagent Co., Ltd.; Dialysis bags: molecular weight cutoff of 8 to 14 kDa, Shanghai Yuanye Biotechnology Co., Ltd.

[0041] Example 1: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, prepared by reacting chitosan with 3-chloro-2-hydroxypropyltrimethylammonium chloride at a molar ratio of 1:3 at 60℃ for 8 h.

[0042] Chitosan quaternary ammonium salt-g-KR-12: The grafting density of KR-12 is 12% of the residual amino group of chitosan quaternary ammonium salt. It is prepared by reacting chitosan quaternary ammonium salt and KR-12 at a molar ratio of 1:0.12 via EDC / NHS coupling reaction at 4℃ for 24h.

[0043] Oxidized sodium alginate: with an oxidation degree of 45%, it is prepared by reacting sodium alginate and sodium periodate in a molar ratio of 1:0.45 at 4°C in the dark for 24 hours.

[0044] Magnesium oxide nanoparticles: the content is 1.5% of the total mass of the hydrogel, and the particle size is 200 to 300 nm.

[0045] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v, soluble in 1% v / v acetic acid solution.

[0046] Oxidized sodium alginate solution: 3% w / v, dissolved in deionized water, containing magnesium oxide nanoparticles, ultrasonically dispersed for 15 min.

[0047] bFGF: The loading amount is 0.03% of the hydrogel mass. The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0048] Preparation process: Step 1: Dissolve 2g of chitosan in 100mL of 1% v / v acetic acid solution, add 9.2g of 3-chloro-2-hydroxypropyltrimethylammonium chloride, stir and react at 60℃ for 8h, adjust the pH to 7.0 with 1mol / L NaOH solution, transfer to a dialysis bag and dialyze in deionized water for 3 days, freeze-dry to obtain chitosan quaternary ammonium salt with a degree of substitution of 0.6.

[0049] Step 2: Dissolve 1g of chitosan quaternary ammonium salt in 100mL of MES buffer at pH 5.5, add 0.23g of EDC and 0.14g of NHS, stir and activate at room temperature for 30min, add 0.15g of KR-12 antimicrobial peptide, stir and react at 4℃ for 24h, transfer to a dialysis bag and dialyze in deionized water for 2 days, freeze-dry to obtain chitosan quaternary ammonium salt-g-KR-12, with KR-12 grafting density of 12% of residual amino groups.

[0050] Step 3: Dissolve 2g of sodium alginate in 100mL of deionized water, add 1.92g of sodium periodate, stir and react for 24h at 4℃ in the dark, add 0.5mL of ethylene glycol to terminate the reaction, transfer to a dialysis bag and dialyze in deionized water for 3 days, freeze dry to obtain oxidized sodium alginate with an oxidation degree of 45%.

[0051] Step 4: Dissolve chitosan quaternary ammonium salt-g-KR-12 in 1% v / v acetic acid solution to prepare a 2.5% w / v solution.

[0052] Sodium alginate was dissolved in deionized water to prepare a 3% w / v solution. 60 mg of magnesium oxide nanoparticles were added and ultrasonically dispersed for 15 min to ensure uniform dispersion of the magnesium oxide nanoparticles.

[0053] Add 0.03% of the bFGF by mass of the hydrogel to a sodium alginate solution containing magnesium oxide nanoparticles and mix thoroughly.

[0054] Step 5: Take 2 mL of chitosan quaternary ammonium salt-g-KR-12 solution and quickly mix it with 2 mL of sodium alginate solution containing magnesium oxide nanoparticles and bFGF. The molar ratio of amino groups of chitosan quaternary ammonium salt to aldehyde groups of sodium alginate is 1:1.0. A hydrogel is formed within 55 seconds to obtain the medical functional dressing.

[0055] The medical functional dressing gel prepared in this embodiment has a gel release time of 55 seconds, a self-healing efficiency of 93%, a liquid absorption capacity of 22 times, a cumulative drug release rate of 32% over 48 hours at pH 7.4, a cumulative drug release rate of 75% over 48 hours at pH 5.5, a structural integrity of 72% over 48 hours at pH 5.5, an antibacterial rate of 99.4% against Escherichia coli, an antibacterial rate of 99.2% against Staphylococcus aureus, an antibacterial rate of 97.5% against MRSA, and a FICI value of 0.30.

[0056] Example 2: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0057] Chitosan quaternary ammonium salt-g-KR-12: KR-12 grafting density is 18% of the residual amino group, and it is prepared by the coupling reaction of chitosan quaternary ammonium salt and KR-12 in a molar ratio of 1:0.18 via EDC / NHS.

[0058] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0059] Magnesium oxide nanoparticles: the content is 1.5% of the total mass of the hydrogel, the same as in Example 1.

[0060] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v.

[0061] Oxidized sodium alginate solution: 3% w / v.

[0062] bFGF: The loading amount is 0.03% of the hydrogel mass. The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0063] Preparation process: Same as the preparation process in Example 1, except that the amount of KR-12 added in step two is adjusted to 0.23g.

[0064] The medical functional dressing gel prepared in this embodiment has a gel release time of 60 seconds, a self-healing efficiency of 91%, a liquid absorption capacity of 21 times, a cumulative drug release rate of 35% over 48 hours at pH 7.4, an cumulative drug release rate of 80% over 48 hours at pH 5.5, a structural integrity of 70% over 48 hours at pH 5.5, an antibacterial rate of 99.6% against Escherichia coli, an antibacterial rate of 99.4% against Staphylococcus aureus, an antibacterial rate of 98.3% against MRSA, and a FICI value of 0.25.

[0065] Example 3: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0066] Chitosan quaternary ammonium salt-g-KR-12: KR-12 grafting density is 6% of the residual amino group, and it is prepared by the coupling reaction of chitosan quaternary ammonium salt and KR-12 in a molar ratio of 1:0.06 via EDC / NHS.

[0067] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0068] Magnesium oxide nanoparticles: the content is 1.5% of the total mass of the hydrogel, the same as in Example 1.

[0069] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v.

[0070] Oxidized sodium alginate solution: 3% w / v. bFGF: loading amount is 0.03% of the hydrogel mass.

[0071] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0072] Preparation process: Same as the preparation process in Example 1, except that the amount of KR-12 added in step two is adjusted to 0.075g.

[0073] The medical functional dressing gel prepared in this embodiment has a gel release time of 50 seconds, a self-healing efficiency of 94%, a liquid absorption capacity of 23 times, a cumulative drug release rate of 25% over 48 hours at pH 7.4, a cumulative drug release rate of 62% over 48 hours at pH 5.5, a structural integrity of 74% over 48 hours at pH 5.5, an antibacterial rate of 99.0% against Escherichia coli, an antibacterial rate of 98.6% against Staphylococcus aureus, an antibacterial rate of 95.5% against MRSA, and a FICI value of 0.38.

[0074] Example 4: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1. Chitosan quaternary ammonium salt-g-KR-12: KR-12 grafting density is 12% of the residual amino group, same as in Example 1.

[0075] Oxidized sodium alginate: with an oxidation degree of 60%, it is prepared by reacting sodium alginate and sodium periodate in a molar ratio of 1:0.6 at 4°C in the dark for 24 hours.

[0076] Magnesium oxide nanoparticles: the content is 1.5% of the total mass of the hydrogel, the same as in Example 1.

[0077] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v.

[0078] Oxidized sodium alginate solution: 3% w / v.

[0079] bFGF: The loading amount is 0.03% of the hydrogel mass.

[0080] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.2.

[0081] Preparation process: Same as the preparation process in Example 1, except that the amount of sodium periodate added in step 3 is adjusted to 2.56g, and the molar ratio of amino to aldehyde in step 5 is adjusted to 1:1.2.

[0082] The medical functional dressing gel prepared in this embodiment has a gel release time of 40 seconds, a self-healing efficiency of 89%, a liquid absorption capacity of 25 times, a cumulative drug release rate of 40% over 48 hours at pH 7.4, a cumulative drug release rate of 82% over 48 hours at pH 5.5, a structural integrity of 68% over 48 hours at pH 5.5, an antibacterial rate of 99.3% against Escherichia coli, an antibacterial rate of 99.1% against Staphylococcus aureus, an antibacterial rate of 97.0% against MRSA, and a FICI value of 0.32.

[0083] Example 5: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0084] Chitosan quaternary ammonium salt-g-KR-12: The grafting density of KR-12 is 12% of the residual amino group, as in Example 1.

[0085] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0086] Magnesium oxide nanoparticles: 2.5% of the total mass of the hydrogel, with a particle size of 200 to 300 nm.

[0087] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v. Oxidized sodium alginate solution: 3% w / v.

[0088] bFGF: The loading amount is 0.05% of the hydrogel mass.

[0089] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0090] Preparation process: Same as the preparation process in Example 1, except that the amount of magnesium oxide nanoparticles added in step four is adjusted to 100 mg.

[0091] The medical functional dressing gel prepared in this embodiment has a gel release time of 45 seconds, a self-healing efficiency of 88%, a liquid absorption capacity of 19 times, a cumulative drug release rate of 30% over 48 hours at pH 7.4, a cumulative drug release rate of 68% over 48 hours at pH 5.5, a structural integrity of 82% over 48 hours at pH 5.5, an antibacterial rate of 99.5% against Escherichia coli, an antibacterial rate of 99.3% against Staphylococcus aureus, an antibacterial rate of 97.2% against MRSA, and a FICI value of 0.31.

[0092] Comparative Example 1: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0093] KR-12 covalent grafting is not performed.

[0094] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0095] It does not contain magnesium oxide nanoparticles.

[0096] Chitosan quaternary ammonium salt solution: 2.5% w / v.

[0097] Oxidized sodium alginate solution: 3% w / v.

[0098] bFGF: The loading amount is 0.03% of the hydrogel mass.

[0099] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0100] Preparation process: Same as the preparation process in Example 1, except that the KR-12 covalent grafting in step two is skipped, magnesium oxide nanoparticles are not added in step four, and ungrafted KR-12 chitosan quaternary ammonium salt is used in step four.

[0101] This approach represents a chitosan quaternary ammonium salt / sodium alginate Schiff base crosslinked hydrogel without antimicrobial peptide covalent grafting and magnesium oxide nanoparticles. It relies solely on the inherent antimicrobial properties of chitosan quaternary ammonium salt and has no pH buffering capacity in acidic environments.

[0102] This comparative example simultaneously removed two variables: KR-12 covalent grafting and magnesium oxide nanoparticles. It was used as a completely blank baseline. Its difference from Example 1 can be evaluated by decomposing it into a single variable comparison with Comparative Example 2 (MgO removed only) and Comparative Example 3 (KR-12 removed only).

[0103] Comparative Example 2: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0104] Chitosan quaternary ammonium salt-g-KR-12: The grafting density of KR-12 is 12% of the residual amino group, as in Example 1.

[0105] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0106] It does not contain magnesium oxide nanoparticles.

[0107] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v.

[0108] Oxidized sodium alginate solution: 3% w / v.

[0109] bFGF: The loading amount is 0.03% of the hydrogel mass.

[0110] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0111] Preparation process: Same as the preparation process in Example 1, except that magnesium oxide nanoparticles are not added in step four.

[0112] This scheme represents a chitosan quaternary ammonium salt-g-KR-12 / sodium alginate Schiff base crosslinked hydrogel with antimicrobial peptide covalent grafts but without magnesium oxide nanoparticles, used to evaluate the contribution of magnesium oxide nanoparticles.

[0113] In acidic infected wounds, excessive hydrolysis of the Schiff base bonds in the hydrogel leads to structural disintegration.

[0114] Comparative Example 3: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0115] KR-12 covalent grafting is not performed.

[0116] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0117] Magnesium oxide nanoparticles: the content is 1.5% of the total mass of the hydrogel, the same as in Example 1.

[0118] Chitosan quaternary ammonium salt solution: 2.5% w / v. Oxidized sodium alginate solution: 3% w / v, containing magnesium oxide nanoparticles.

[0119] bFGF: The loading amount is 0.03% of the hydrogel mass.

[0120] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0121] Preparation process: Same as in Example 1, except for step two, KR-12 covalent grafting.

[0122] This scheme represents a chitosan quaternary ammonium salt / sodium alginate / MgO Schiff base crosslinked hydrogel with magnesium oxide nanoparticles but without antimicrobial peptide covalent grafting, used to evaluate the contribution of KR-12 covalent grafting.

[0123] Comparative Example 4: Raw material formula: Chitosan: Deacetylation degree 90% to 95%, without quaternization modification.

[0124] KR-12 antimicrobial peptide: physically mixed loading, non-covalent grafting, the amount added is the same as the grafting amount in Example 1.

[0125] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0126] It does not contain magnesium oxide nanoparticles.

[0127] Chitosan solution: 2.5% w / v.

[0128] Oxidized sodium alginate solution: 3% w / v, KR-12 is added directly to the oxidized sodium alginate solution.

[0129] bFGF: The loading amount is 0.03% of the hydrogel mass.

[0130] The molar ratio of chitosan amino groups to sodium alginate aldehyde groups is 1:1.0.

[0131] Preparation process: Same as the preparation process in Example 1, skipping the quaternization modification in step one and the covalent grafting in step two. In step four, KR-12 is directly physically mixed in the sodium alginate oxidase solution without adding magnesium oxide nanoparticles.

[0132] This scheme represents the existing technical route of physically loading KR-12 by cross-linking unquaternized chitosan and oxidized sodium alginate Schiff base, corresponding to the scheme in CN119901234A.

[0133] This comparative example differs from Example 1 in several ways (no quaternization, physical mixing of KR-12 instead of covalent grafting, and no magnesium oxide), and is a comprehensive comparison of different technical routes rather than a single-variable comparison.

[0134] Comparative Example 5: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.5, prepared by reacting chitosan with 3-chloro-2-hydroxypropyltrimethylammonium chloride at a molar ratio of 1:2.5 at 60℃ for 8 h.

[0135] Oxidized sodium alginate: with an oxidation degree of 55%, it is prepared by reacting sodium alginate and sodium periodate in a molar ratio of 1:0.55 at 4°C in the dark for 24 hours.

[0136] It does not contain KR-12 antimicrobial peptide.

[0137] It does not contain magnesium oxide nanoparticles.

[0138] No bFGF load.

[0139] Chitosan quaternary ammonium salt solution: 2.5% w / v.

[0140] Oxidized sodium alginate solution: 3% w / v.

[0141] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.1.

[0142] Preparation process: Chitosan quaternary ammonium salt solution and sodium alginate oxidized solution are rapidly mixed at a volume ratio of 1:1 to form a hydrogel within 65 seconds.

[0143] Other details: This solution represents the existing technology of oxidized sodium alginate / chitosan quaternary ammonium salt self-healing hydrogel, corresponding to the solution in CN119789012A. It relies solely on the inherent antibacterial properties of chitosan quaternary ammonium salt and contains no antimicrobial peptides, magnesium oxide, or growth factors. This comparative example differs from Example 1 in several ways (it does not contain KR-12, MgO, or bFGF), and represents a comprehensive comparison of different technical routes rather than a single-variable comparison.

[0144] Comparative Example 6: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0145] Chitosan quaternary ammonium salt-g-KR-12: The grafting density of KR-12 is 12% of the residual amino group, as in Example 1.

[0146] Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0147] Calcium oxide nanoparticles: the content is 1.5% of the total mass of the hydrogel, the particle size is 200 to 300 nm, and they replace magnesium oxide nanoparticles.

[0148] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v. Oxidized sodium alginate solution: 3% w / v, containing calcium oxide nanoparticles.

[0149] bFGF: The loading amount is 0.03% of the hydrogel mass.

[0150] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0151] Preparation process: Same as the preparation process in Example 1, except that in step four, an equal amount of calcium oxide nanoparticles are used to replace magnesium oxide nanoparticles.

[0152] This scheme represents a control scheme that uses calcium oxide nanoparticles instead of magnesium oxide nanoparticles. The calcium ions released from the dissolution of calcium oxide form strong eggshell-like ionic crosslinks with sodium alginate, resulting in an excessively dense hydrogel.

[0153] Comparative Example 7: Raw material formula: Chitosan quaternary ammonium salt: degree of substitution 0.6, same as in Example 1.

[0154] Chitosan quaternary ammonium salt-g-KR-12: KR-12 grafting density is 12% of the residual amino group, same as in Example 1. Oxidized sodium alginate: oxidation degree 45%, same as in Example 1.

[0155] Magnesium chloride: The amount added is the same as the molar amount of magnesium oxide nanoparticles in Example 1, replacing the magnesium oxide nanoparticles.

[0156] Chitosan quaternary ammonium salt-g-KR-12 solution: 2.5% w / v.

[0157] Oxidized sodium alginate solution: 3% w / v, containing magnesium chloride.

[0158] bFGF: The loading amount is 0.03% of the hydrogel mass.

[0159] The molar ratio of amino groups in chitosan quaternary ammonium salt to aldehyde groups in sodium alginate is 1:1.0.

[0160] Preparation process: Same as the preparation process in Example 1, except that magnesium oxide nanoparticles are replaced with magnesium chloride in an equal molar amount of magnesium in step four.

[0161] This approach represents a control scheme that replaces magnesium oxide nanoparticles with soluble magnesium salts. Magnesium chloride dissolves immediately, releasing all magnesium ions, has no pH buffering capacity, and the magnesium ions cause rapid ionic cross-linking of sodium alginate oxide.

[0162] To enable those skilled in the art to clearly understand the technical solution of this invention, completely replicate the preparation process and performance testing experiments, and implement this invention without creative effort, the abbreviations, technical terms, definitions of key parameters, metrological standards, general detailed conditions for the preparation process, and basic experimental conditions for performance testing involved in the entire text are uniformly explained as follows.

[0163] All abbreviations and codes used in this invention are common identifiers in the fields of biomedicine and polymer materials, and their specific meanings are as follows: g-KR-12 represents the grafting product of chitosan quaternary ammonium salt covalently grafted with KR-12 antimicrobial peptide; EDC is 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride, and NHS is N-hydroxysuccinimide. The combination of the two is a classic activating agent for the amidation coupling of peptides and polysaccharide amino groups; MES is 2-morpholinoethanesulfonic acid, which is the buffer medium used in the grafting reaction of the antimicrobial peptide in this invention; bFGF is basic fibroblast growth factor, which is the active protein loaded in this dressing that promotes wound healing; MRSA is methicillin-resistant Staphylococcus aureus, which is the drug-resistant pathogen targeted by this invention; FICI is the graded inhibitory concentration index, used to evaluate the synergistic effect of two antimicrobial substances; w / v represents the mass-volume percentage concentration, that is, the mass (g) of solute contained in 100 mL of solution; CFU is colony-forming unit, which is the standard unit for counting viable microorganisms.

[0164] At the same time, the standard name of the chemical substance of this invention is corrected: = Chitosan glycidyltrimethylammonium chloride is standardly named hydroxypropyltrimethylammonium chloride modified chitosan (chitosan quaternary ammonium salt), which is a quaternized derivative obtained by alkaline ring-opening reaction of chitosan and 3-chloro-2-hydroxypropyltrimethylammonium chloride.

[0165] In this invention, the content and functional parameters of all components are clearly defined and uniformly measured by a unified standard and calculation rule. All content and loading indicators are measured by the total mass of the wet composite hydrogel after molding.

[0166] The degree of substitution of chitosan quaternary ammonium salt is defined as the percentage of the number of moles of active amino groups on the chitosan molecular chain that are replaced by quaternary ammonium cationic groups, relative to the total number of moles of amino groups in chitosan. In this invention, the degree of substitution ranges from 0.4 to 0.8, corresponding to a molar substitution percentage of 40% to 80%. The covalent grafting density of KR-12 antimicrobial peptide is defined as the percentage of the number of moles of KR-12 antimicrobial peptide grafted onto the residual amino groups of chitosan quaternary ammonium salt, relative to the total number of moles of residual amino groups in chitosan quaternary ammonium salt. In this invention, the grafting density ranges from 5% to 20%, all in molar percentage. The oxidation degree of oxidized sodium alginate is defined as the percentage of the number of moles of the vicinal diol structure in the sodium alginate molecular chain. The percentage of monosaccharide units oxidized to aldehydes by sodium periodate relative to the total number of monosaccharide units in sodium alginate, with an oxidation range of 30%-60% corresponding to the percentage of aldehyde-modified monosaccharide units; the dressing structural integrity refers to the percentage ratio of the remaining mass of the sample after immersion in an acidic environment to the mass of the control sample; the self-healing efficiency is the percentage ratio of the storage modulus of the hydrogel after self-healing to the storage modulus before self-healing; the cumulative drug release rate specifically refers to the percentage of the cumulative release of KR-12 antimicrobial peptides over 48 hours under different pH conditions relative to the total load. The calculation logic for the above three indicators is uniformly applicable to all embodiments and comparative examples of this invention. The dressing absorbance is the ratio of the saturated wet weight of the lyophilized dry gel after absorbing PBS buffer to the initial weight of the dry gel, and is a dimensionless multiple indicator.

[0167] For the general process parameters, reagent ratios and post-treatment conditions not detailed in the preparation method of this invention, we now provide a unified and detailed supplement to ensure that the technical solution can be stably reproduced across the entire range.

[0168] First, the preparation process of chitosan quaternary ammonium salt: The solid-liquid ratio of the reaction system of chitosan and 3-chloro-2-hydroxypropyltrimethylammonium chloride is uniformly controlled at 2g / 100mL. After the reaction, the pH of the system is adjusted to neutral (pH=7.0) with 1mol / L sodium hydroxide solution. Dialysis is performed using a dialysis bag with a molecular weight cutoff of 8-14kDa. Dialysis is performed continuously with deionized water at room temperature for 3 days, and the dialysate is changed twice a day. Finally, solid chitosan quaternary ammonium salt is obtained by freeze drying. This process is suitable for preparing products with a degree of substitution of 0.4-0.8.

[0169] Second, the covalent grafting process of KR-12 antimicrobial peptide EDC / NHS: the standard molar ratio of chitosan quaternary ammonium salt, EDC, and NHS is 1:0.25:0.15. During the activation stage, the mixture is stirred at room temperature for 30 minutes. MES buffer with pH 5.5-6.0 is used throughout the grafting reaction. After the reaction, unreacted small molecules are removed by dialysis at room temperature for 2 days using a dialysis bag with a molecular weight cutoff of 8-14 kDa. Then, the mixture is freeze-dried to obtain chitosan quaternary ammonium salt-g-KR-12.

[0170] Third, the preparation process of oxidized sodium alginate: Sodium alginate and sodium periodate react in the dark throughout the process and are stirred at a constant temperature of 4°C. When the reaction is terminated, 0.5% by volume of ethylene glycol is added according to the volume of the reaction system to quench the remaining sodium periodate. After quenching, stirring is continued for 30 minutes. Then, dialysis is performed at room temperature for 3 days using a dialysis bag with a molecular weight cutoff of 8-14 kDa. After freeze drying, oxidized sodium alginate with an oxidation degree of 30%-60% is obtained.

[0171] Fourth, the inorganic nanoparticle dispersion process: After adding magnesium oxide / calcium oxide nanoparticles to the sodium alginate aqueous solution, they are dispersed using conventional laboratory ultrasonic equipment with an ultrasonic power of 200W and an ultrasonic duration of 15min to ensure that the nanoparticles are uniformly dispersed in the system; magnesium chloride can be dissolved directly by stirring without ultrasonic treatment.

[0172] Fifth, preparation of hydrogel precursor solution: Chitosan quaternary ammonium salt-g-KR-12 is prepared into a 2%-4% w / v solution using a 1% volume fraction glacial acetic acid aqueous solution, and sodium alginate is prepared into a 2%-5% w / v solution using deionized water. The two precursor solutions are rapidly mixed at a volume ratio of 1:1 to initiate the Schiff base crosslinking reaction.

[0173] All performance testing experiments in this invention adopted uniform basic environmental conditions. All in vitro tests were conducted at a constant temperature of 37°C to simulate the physiological temperature of a human wound. The pH 7.4 and pH 5.5 test media were standard phosphate-buffered saline (PBS) with an ionic strength of 0.1 mol / L. The initial concentration of the bacterial solution used in the antibacterial experiments was uniformly 1 × 10⁻⁶. 6 CFU / mL, bacterial culture was conducted using conventional nutrient agar medium; cytotoxicity experiments used L929 mouse fibroblasts, and hydrogel extracts were prepared according to national standard medical material extraction methods; animal wound healing experiments used SPF-grade SD rats to construct full-thickness skin defects with a diameter of 1.5 cm, and the experimental environment was kept at constant temperature and humidity; sampling, fluid replenishment, and weighing procedures for in vitro experiments such as rheology, swelling, and drug release were standardized to ensure cross-sectional comparability of test data for different samples.

[0174] Performance testing methods and results analysis: (1) Gel time: The test tube inversion method was used. After mixing the two solutions, the timing was started. The test tube did not flow within 30 seconds of being inverted, indicating that it had gelled. The gel time was recorded.

[0175] (2) Self-healing efficiency: The hydrogel was cut into two halves and placed together. After being placed at 37°C for 2 hours, a rheological test was performed. The storage modulus Gx before and after self-healing was recorded. The self-healing efficiency is equal to Gx after self-healing divided by Gx before self-healing multiplied by 100%.

[0176] (3) Liquid absorption: Weigh the lyophilized hydrogel and record it as W0. Immerse it in PBS buffer at pH 7.4 and soak it at 37°C for 24 hours. After soaking, remove it and absorb the surface moisture with filter paper. Weigh it and record it as W1. The liquid absorption is equal to W1 divided by W0.

[0177] (4) Drug release rate: The hydrogel containing KR-12 was immersed in 10 mL of PBS buffer at pH 7.4 or pH 5.5 and shaken at 37°C. 1 mL samples were taken at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h and 48 h respectively, and an equal amount of fresh PBS was added. The concentration of KR-12 was determined by HPLC and the cumulative drug release rate was calculated.

[0178] (5) Structural integrity: The hydrogel was immersed in 20 mL of pH 5.5 PBS buffer and placed at 37°C for 48 h. After that, it was taken out and the surface moisture was gently absorbed with filter paper. The weight was recorded as Wt. At the same time, the same mass of hydrogel was immersed in pH 7.4 PBS buffer for 48 h and the weight was recorded as Wn. The structural integrity was equal to Wt divided by Wn multiplied by 100%.

[0179] (6) Antibacterial rate: The colony counting method was used. The hydrogel sample was co-cultured with culture medium containing 10^6 CFU / mL of bacterial solution at 37℃ for 24h. The culture solution was spread on plates and counted. The antibacterial rate was equal to the number of colonies in the control group minus the number of colonies in the experimental group, divided by the number of colonies in the control group, and multiplied by 100%.

[0180] (7) FICI value: The checkerboard dilution method was used to dilute chitosan quaternary ammonium salt and KR-12 by 2-fold serial dilution. After combination, they were co-cultured with MRSA for 24h and the MIC was measured. FICI is equal to the combined MIC of chitosan quaternary ammonium salt divided by the MIC of chitosan quaternary ammonium salt alone plus the combined MIC of KR-12 divided by the MIC of KR-12 alone.

[0181] FICI less than or equal to 0.5 indicates synergy, 0.5 less than FICI less than or equal to 1.0 indicates addition, 1.0 less than FICI less than or equal to 4.0 indicates indifference, and FICI greater than 4.0 indicates antagonism.

[0182] (8) Cytotoxicity: L929 fibroblasts were co-cultured with hydrogel extract for 24 h and 48 h using the CCK-8 method, and cell viability was determined.

[0183] (9) Wound healing rate: A full-thickness skin defect model of SD rats was established with a wound diameter of 1.5 cm. Each experimental group was covered with dressings. The wound area was measured at 3d, 7d and 14d. The wound healing rate was equal to the initial wound area minus the wound area at each time point, divided by the initial wound area and multiplied by 100%.

[0184] (10) Histological evaluation: wound tissue was taken for HE staining and Masson trichrome staining to assess the degree of epithelialization and the amount of collagen deposition.

[0185] Test results: Table 1. Test results of the physicochemical properties and drug release performance of the hydrogel:

[0186] Test methods: gelation time was measured using the inverted test tube method; self-healing efficiency was measured using rheological tests; the liquid absorption volume was determined by soaking in PBS for 24 hours; the drug release rate was determined by HPLC as the cumulative release rate over 48 hours; the drug release rate ratio was calculated as the drug release rate at pH 5.5 divided by the drug release rate at pH 7.4; and structural integrity was determined by the mass retention rate after soaking in PBS at pH 5.5 for 48 hours.

[0187] Results Analysis: Examples 1 to 5 had gel times of 40 to 60 seconds, self-healing efficiencies of 88% to 94%, liquid absorption of 19 to 25 times, drug release rates of 25% to 40% at pH 7.4, and 62% to 82% at pH 5.5, with a release rate ratio of 2.05 to 2.48. The structural integrity at pH 5.5 was 68% to 82%, exhibiting both pH-responsive drug release characteristics and structural integrity under acidic conditions. Example 5 had the highest magnesium oxide content (2.5%) and the highest structural integrity at pH 5.5 (82%), but the drug release rate decreased to 68% and the liquid absorption decreased to 19 times, indicating that excessive magnesium oxide nanoparticles made the hydrogel network too dense, affecting drug diffusion and liquid absorption performance. Example 4 had a higher oxidation degree (60%), higher aldehyde content, and higher crosslinking density, with the shortest gel time (40 seconds) and a relatively lower structural integrity at pH 5.5 (68%). This indicates that the high Schiff base bond density at high oxidation degrees leads to a large total hydrolysis at pH 5.5, and even with magnesium oxide buffering, the structural integrity remained slightly low.

[0188] Comparative Example 1, without KR-12 grafting and containing no magnesium oxide, had a drug release rate of 0% and a structural integrity of only 33% at pH 5.5. This indicates that the inherent antibacterial properties of chitosan quaternary ammonium salts do not produce a detectable drug release signal, and that without magnesium oxide buffering, excessive hydrolysis of Schiff base bonds at pH 5.5 leads to severe structural disintegration. Comparative Example 2, with KR-12 covalent grafting but without magnesium oxide, had a drug release rate of 82% at pH 5.5, close to the 75% of Example 1. However, its structural integrity at pH 5.5 was only 28%, far lower than the 72% of Example 1. This indicates that without magnesium oxide buffering, the hydrogel's structure severely disintegrates in an acidic environment. Although the drug release rate appears high, this is an uncontrolled release caused by structural disintegration rather than a smart responsive release. This result demonstrates that the pH buffering mechanism of magnesium oxide nanoparticles is crucial for maintaining the structural integrity of hydrogels in acidic environments, solving the technical problem of the incompatibility between pH-responsive drug release and structural integrity.

[0189] Comparative Example 3 contained magnesium oxide but lacked KR-12 covalent grafting. Its structural integrity at pH 5.5 was 76%, close to the 72% of Example 1, indicating that the pH buffering effect of magnesium oxide is independent of the presence of KR-12. However, the drug release rate was 0, showing no pH-responsive drug release capability. Its antibacterial rate against MRSA was only 88.2%, far lower than the 97.5% of Example 1, demonstrating that relying solely on the inherent antibacterial properties of chitosan quaternary ammonium salt and the buffering effect of magnesium oxide is insufficient to achieve long-lasting antibacterial effects.

[0190] Comparative Example 4, with KR-12 physically loaded and without quaternized chitosan or magnesium oxide, showed a release rate as high as 65% at pH 7.4, indicating severe burst release. At pH 5.5, the structural integrity was only 22%, and the release rate ratio was only 1.35, suggesting that physically loaded KR-12 exhibited rapid release at pH 7.4 with insignificant pH response characteristics. Comparative Example 5, simulating CN119789012A, only involved chitosan quaternary ammonium salt / oxidized sodium alginate Schiff base crosslinking without KR-12 or magnesium oxide. At pH 5.5, the structural integrity was 30%, and the antibacterial rate against MRSA was 85.0%.

[0191] Comparative Example 6, which replaced magnesium oxide with calcium oxide, showed a gel time of only 15 seconds, a self-healing efficiency of only 45%, a liquid absorption of only 8 times, a drug release rate of only 12% at pH 7.4, and a drug release rate of only 18% at pH 5.5. The calcium ions released from the dissolution of calcium oxide rapidly formed strong eggshell-like ionic cross-links with sodium alginate, making the hydrogel excessively dense and rigid. Although the structural integrity reached 92% at pH 5.5, almost no drug could be released, thus losing the basic function of a functional dressing.

[0192] This result proves that magnesium oxide nanoparticles cannot be replaced by calcium oxide. The weak ionic crosslinking formed between magnesium oxide and sodium alginate maintains structural integrity without affecting drug release, while the strong ionic crosslinking of calcium oxide causes the hydrogel to lose its drug release and liquid absorption functions. Although both are basic oxides, their effects are fundamentally different. The uniqueness of magnesium oxide lies in the fact that the magnesium ions produced by its dissolution form a weak ionic crosslinking with sodium alginate, rather than a strong eggshell structure.

[0193] Comparative Example 7, which replaced magnesium oxide nanoparticles with magnesium chloride, had a gelation time of 35 seconds, a pH of 5.5, and a structural integrity of 42%, significantly lower than the 72% in Example 1. Magnesium chloride dissolved immediately, releasing all magnesium ions, and had no pH buffering capacity. As a neutral salt, magnesium chloride neither consumes hydrogen ions nor releases hydroxide ions, thus failing to buffer the pH value of the hydrogel microenvironment.

[0194] Furthermore, the magnesium ions dissolved by magnesium chloride cause rapid ionic cross-linking of sodium alginate oxide, resulting in a denser hydrogel with a liquid absorption capacity 16 times lower than that of Example 1 (22 times lower). This result demonstrates that magnesium oxide nanoparticles cannot be replaced by soluble magnesium salts. The core function of magnesium oxide nanoparticles lies in their gradual dissolution under acidic conditions, consuming hydrogen ions and releasing hydroxide ions to provide a pH buffering effect, while magnesium chloride only provides magnesium ions and does not provide a pH buffer.

[0195] Table 2. Results of antibacterial properties and wound healing properties tests:

[0196] Test methods: Antibacterial rate was measured by colony counting method over 24 hours; MRSA was measured by checkerboard dilution method using FICI; and wound healing rate was measured using a full-thickness skin defect model in SD rats.

[0197] Results Analysis: Examples 1 to 5 showed antibacterial rates of 99.0% to 99.6% against *Escherichia coli*, 98.6% to 99.4% against *Staphylococcus aureus*, and 95.5% to 98.3% against *MRSA*, with FICI values ​​ranging from 0.25 to 0.38, all indicating significant synergistic effects. Example 2, with the highest grafting density of 18%, showed the highest *MRSA* antibacterial rate of 98.3% and the lowest FICI value of 0.25, demonstrating the strongest synergistic effect. Example 3, with the lowest grafting density of 6%, showed a *MRSA* antibacterial rate of 95.5%, and a FICI value of 0.38, still within the range of significant synergistic effects.

[0198] Comparative Example 1, without KR-12 grafting and magnesium oxide, showed an MRSA antibacterial rate of only 84.5%, far lower than the 97.5% of Example 1, indicating that relying solely on the inherent antibacterial properties of chitosan quaternary ammonium salt is insufficient for the effectiveness of MRSA. Comparative Example 2, with KR-12 covalent grafting but without magnesium oxide, achieved an MRSA antibacterial rate of 96.8%, close to the 97.5% of Example 1, and a similar FICI value of 0.32, indicating that the addition of magnesium oxide nanoparticles does not affect the antibacterial performance itself. However, the 7-day wound healing rate of only 52% was far lower than the 63% of Example 1, and the 14-day wound healing rate of 85% was far lower than the 96% of Example 1. This is because the hydrogel's structure disintegrates under acidic infected wound conditions, losing its barrier function and failing to maintain a moist environment and sustain drug release.

[0199] This result demonstrates that magnesium oxide nanoparticles indirectly promote wound healing by maintaining the structural integrity of the hydrogel, and the synergistic effect of antibacterial properties and structural stability is reflected in the wound healing rate rather than the antibacterial rate itself.

[0200] Comparative Example 3 contained magnesium oxide but lacked KR-12 covalent grafting. The MRSA antibacterial rate was 88.2%, the wound healing rate was 50% at 7 days, and 84% at 14 days. Although the hydrogel structure was intact, the insufficient antibacterial effect led to persistent wound infection, and the healing rate was lower than that of Example 1.

[0201] Compared to the control group 4, the MRSA antibacterial rate was 86.5%, and the FICI value was 0.62, indicating an additive rather than synergistic effect. The wound healing rate was 42% at 7 days and 75% at 14 days. KR-12 physical mixing loading resulted in severe burst release, with 65% released within 24 hours and the concentration rapidly decreasing. Furthermore, the hydrogel structure disintegrated in an acidic environment. These two factors combined led to the lowest wound healing rate.

[0202] Compared with the control group 5, the antibacterial rate of MRSA was 85.0%, the wound healing rate at 7 days was 45%, and the wound healing rate at 14 days was 79%.

[0203] Comparative Example 6, which replaced magnesium oxide with calcium oxide, showed an MRSA antibacterial rate of 96.5%, similar to Example 1. However, the wound healing rate was only 48% at 7 days and 82% at 14 days. This was because the hydrogel was too dense and released almost no drug, with bFGF and KR-12 locked in the dense network and unable to reach the wound.

[0204] This result demonstrates that the antibacterial rate and wound healing rate are not simply proportional. The antibacterial rate reflects the bactericidal ability, while the wound healing rate also depends on whether the drug can be effectively delivered to the wound. Although calcium oxide made the hydrogel dense and maintained the antibacterial rate, it hindered drug release, resulting in a significantly lower wound healing rate than in Example 1.

[0205] Comparative Example 7, which replaced magnesium oxide nanoparticles with magnesium chloride, showed an MRSA antibacterial rate of 95.8%, a wound healing rate of 55% at 7 days, and a wound healing rate of 88% at 14 days, all lower than the 63% and 96% of Example 1, respectively. This is because the hydrogel's structural integrity was only 42% in an acidic environment. Although magnesium chloride provides magnesium ions to promote angiogenesis, it lacks pH buffering capacity, and the hydrogel still partially disintegrated, affecting the sustained release of the drug.

[0206] Based on the 52% wound healing rate of Comparative Example 2 (without magnesium oxide) and the 50% wound healing rate of Comparative Example 3 (without KR-12), the simple sum of the increases in these two methods was expected to reach approximately 55% for Example 1. However, Example 1 actually achieved a 63% wound healing rate after 7 days, exceeding the expected sum by 15%. Regarding the 14-day wound healing rate, Comparative Example 2 achieved 85%, and Comparative Example 3 achieved 84%, with a simple sum expected of approximately 86%. Example 1, however, achieved 96%, exceeding the expected sum by 12%. This synergistic effect, exceeding the simple sum, stems from the magnesium oxide nanoparticles maintaining structural integrity, enabling sustained release of KR-12 rather than a one-time release due to structural disintegration, and the synergistic effect of magnesium ions promoting angiogenesis and bFGF promoting fibroblast proliferation. Regarding the FICI value, Comparative Example 4 had a value of 0.62, which is additive, while Example 1 had a value of 0.30, which is significantly synergistic. This demonstrates that the inherent antibacterial properties of chitosan quaternary ammonium salt, KR-12 covalent grafting, and magnesium oxide nanoparticles work synergistically to produce antibacterial and wound healing enhancements that exceed the simple sum of their individual effects.

[0207] In summary, this invention achieves stable hydrogel structure, intelligent drug release, and long-lasting antibacterial effect in acidic infected wounds through the synergistic combination of three mechanisms: self-regulating pH buffering of magnesium oxide nanoparticles, anti-surge release by covalent grafting of antimicrobial peptides, and dual antimicrobial synergy. There is a strict synergistic relationship between the various technical features, which cannot be obtained without creative labor.

[0208] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A medical functional dressing, characterized in that, The invention includes a dynamic cross-linked hydrogel composed of chitosan quaternary ammonium salt-g-KR-12 / sodium alginate oxide / magnesium oxide nanoparticles. The dynamic cross-linked network is formed by covalently grafting KR-12 antimicrobial peptides onto chitosan quaternary ammonium salt and sodium alginate oxide through a Schiff base reaction. Magnesium oxide nanoparticles are uniformly dispersed in the hydrogel network and simultaneously loaded with basic fibroblast growth factor bFGF. The degree of substitution of the chitosan quaternary ammonium salt is 0.4 to 0.8; the amino acid sequence of the KR-12 antimicrobial peptide is KRIVQRIKDFLR, and the covalent grafting density is 5% to 20% of the residual amino groups of the chitosan quaternary ammonium salt; The degree of oxidation of the sodium alginate is 30% to 60%; the content of the magnesium oxide nanoparticles is 0.5% to 3% of the total mass of the hydrogel, and the particle size is 100 to 500 nm; the molar ratio of the amino group of the chitosan quaternary ammonium salt to the aldehyde group of the sodium alginate is 1:0.8 to 1:1.

5.

2. The medical functional dressing as described in claim 1, characterized in that, The chitosan quaternary ammonium salt is chitosan glycidyltrimethylammonium chloride, which is prepared by reacting chitosan with 3-chloro-2-hydroxypropyltrimethylammonium chloride under alkaline conditions.

3. The medical functional dressing as described in claim 1, characterized in that, The KR-12 antimicrobial peptide was covalently grafted onto the residual amino group of chitosan quaternary ammonium salt via an EDC / NHS coupling reaction. The grafting reaction was carried out in MES buffer at pH 5.5 to 6.0, at a reaction temperature of 4°C, and for a reaction time of 24 h.

4. The medical functional dressing as described in claim 1, characterized in that, The oxidized sodium alginate is prepared by oxidizing sodium alginate and sodium periodate under light-protected conditions. The molar ratio of sodium alginate to sodium periodate is 1:0.3 to 1:0.6, the oxidation reaction temperature is 4°C, and the reaction time is 24h.

5. The medical functional dressing as described in claim 1, characterized in that, The magnesium oxide nanoparticles dissolve and release hydroxide ions and magnesium ions in an acidic infected wound environment. The hydroxide ions locally buffer the pH value of the hydrogel microenvironment, inhibiting excessive hydrolysis of Schiff base bonds and maintaining the integrity of the hydrogel structure. The magnesium ions form weak ionic crosslinks with the carboxyl groups of oxidized sodium alginate, partially compensating for the decrease in crosslink density caused by the hydrolysis of Schiff base bonds, thus achieving a synergistic effect of accelerated drug release and structural stability in infected wounds.

6. The medical functional dressing as described in claim 1, characterized in that, The bFGF is loaded at a rate of 0.01% to 0.05% of the hydrogel mass and is loaded into the hydrogel network through physical adsorption.

7. The medical functional dressing as described in claim 1, characterized in that, The medical functional dressing has a structural integrity of not less than 65% at pH 5.5 for 48 hours, a cumulative drug release rate of 25% to 45% at pH 7.4 for 48 hours, and a cumulative drug release rate of 60% to 85% at pH 5.5 for 48 hours.

8. The medical functional dressing as described in claim 1, characterized in that, The medical functional dressing has an antibacterial rate of not less than 99% against Escherichia coli, not less than 98% against Staphylococcus aureus, and not less than 95% against methicillin-resistant Staphylococcus aureus (MRSA).

9. The medical functional dressing as described in claim 1, characterized in that, The gel time of this medical functional dressing is 30 to 120 seconds, the self-healing efficiency is no less than 85%, and the liquid absorption capacity is 15 to 30 times its own weight.

10. A method for preparing a medical functional dressing according to any one of claims 1 to 9, characterized in that, Includes the following steps: Step 1: Chitosan is reacted with 3-chloro-2-hydroxypropyltrimethylammonium chloride at 60°C for 8 hours to obtain chitosan quaternary ammonium salt; Step 2: KR-12 antimicrobial peptide is covalently grafted onto the residual amino group of chitosan quaternary ammonium salt via EDC / NHS coupling reaction to obtain chitosan quaternary ammonium salt-g-KR-12. Step 3: Sodium alginate and sodium periodate are reacted at 4°C in the dark for 24 hours to obtain oxidized sodium alginate. Step 4: Dissolve chitosan quaternary ammonium salt-g-KR-12 in acetic acid solution to prepare a 2% to 4% w / v solution; dissolve sodium alginate in deionized water to prepare a 2% to 5% w / v solution; ultrasonically disperse magnesium oxide nanoparticles in sodium alginate solution to make the magnesium oxide content 0.5% to 3% of the total mass of hydrogel; add bFGF to the sodium alginate solution containing magnesium oxide nanoparticles and mix evenly. Step 5: Quickly mix the chitosan quaternary ammonium salt-g-KR-12 solution with the sodium alginate oxidized solution containing magnesium oxide nanoparticles and bFGF at a volume ratio of 1:

1. The molar ratio of the amino group of the chitosan quaternary ammonium salt to the aldehyde group of the sodium alginate oxidized solution is 1:0.8 to 1:1.

5. A hydrogel is formed within 30 to 120 seconds to obtain the medical functional dressing.

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