A method for preparing a bactericidal hemostatic medical dressing

Abstract

The present application relates to the technical field of medical biomaterials, and discloses a preparation method of a sterilization and hemostasis medical dressing, which comprises the following steps: preparing solution A containing a cationic polymer; dissolving an anionic polysaccharide, a plant polyphenol and a divalent cation source in water, and adjusting the pH value to 4.5-6.0 to prepare solution B. Solution A is mixed with solution B to form a liquid precursor, and the pH value of the precursor increases after contacting a physiological pH environment, which triggers in-situ gelation. The gelation is quickly completed through the synergistic effect of electrostatic attraction between the cationic polymer and the anionic polysaccharide and the plant polyphenol, coordination crosslinking between the divalent cation and each polymer, and a hydrogen bond network. The dressing prepared by the present application can quickly gel in-situ, closely adhere to a wound surface, has efficient sterilization and hemostasis functions, and can be removed on demand and without damage through an unlocking liquid containing a chelating agent.

Description

Technical Field

[0001] This invention relates to the field of medical biomaterials technology, and in particular to a method for preparing a bactericidal and hemostatic medical dressing. Background Technology

[0002] Wound dressings are crucial medical supplies used clinically to cover wounds, absorb exudate, prevent infection, and promote tissue healing. Traditional wound dressings, such as gauze and cotton pads, while widely used, have inherent limitations. Their fixed shape makes it difficult to achieve a perfect fit to irregular or deep wound contours, often leaving gaps that provide conditions for bacterial growth. Furthermore, traditional dressings tend to adhere to newly formed granulation tissue during dressing changes, and their removal is often accompanied by severe pain and may cause secondary mechanical damage to the fragile healing tissue.

[0003] To overcome the aforementioned drawbacks, hydrogels have attracted widespread attention as a novel medical dressing. Among them, dressing systems that can directly undergo phase transition at the wound site from liquid precursors to form gels in situ have become a research hotspot due to their excellent tissue adhesion. However, existing in-situ gel dressing technologies still face challenges. The gelation process of many systems is difficult to control precisely. For example, the gelation reaction triggered by simply mixing two precursor solutions has an uncontrollable onset time, resulting in an excessively short operating window before application and difficulty in ensuring gel uniformity.

[0004] Furthermore, for treating complex wounds that are bleeding or prone to infection, ideal dressings should not only provide a physical barrier but also possess active biological functions. Many existing hydrogel dressings perform well in providing a moist healing environment, but they fall short in integrating highly effective bactericidal capabilities and rapid hemostasis, making it difficult to meet the comprehensive treatment needs of complex wounds.

[0005] More importantly, the problem of non-invasive removal of dressings remains unresolved. To ensure sufficient mechanical strength, many in-situ formed gels rely on stable chemical cross-linking networks, making them difficult to remove once formed. During dressing change cycles, removing these irreversible gel dressings, like traditional dressings, still carries the risk of damaging newly formed tissue, violating the principles of non-invasive treatment.

[0006] Therefore, developing a novel in-situ gel dressing preparation method that allows for controllable gelation, perfect conformity to wound contours, integration of efficient sterilization and hemostasis, and ultimately on-demand, non-invasive removal is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] The technical problem to be solved by this application is to provide a method for preparing a medical dressing that can be rapidly gelled in situ, has both bactericidal and hemostatic functions, and can be removed on demand without damage.

[0008] To solve the above-mentioned technical problems, the present invention provides a method for preparing a bactericidal and hemostatic medical dressing, which adopts the following technical solution:

[0009] A method for preparing a bactericidal and hemostatic medical dressing includes the following steps:

[0010] S1. Prepare solution A, wherein solution A is an aqueous solution containing a cationic polymer;

[0011] S2. Prepare solution B, wherein step S2 includes dissolving anionic polysaccharides, plant polyphenols and divalent cation sources in an aqueous solution and adjusting the pH of solution B to 4.5-6.0;

[0012] S3. Mix the solution A and the solution B to form a liquid precursor that can gel in situ upon contact with a physiological pH environment, thereby obtaining the medical dressing.

[0013] By employing the above technical solution, the dressing precursor is packaged into two independent solutions. In step S2, the pH of solution B is preset within the acidic range (4.5–6.0). This inhibits premature interactions between components within the system, particularly suppressing the deprotonation of functional groups in anionic polysaccharides and plant polyphenols, thus ensuring the fluidity and stability of the liquid precursor before application. When this liquid precursor is applied to the wound, a rapid pH change occurs at the interface between it and the wound tissue and exudate. Specifically, the wound exudate, as a strong buffering system containing bicarbonate and phosphate, can rapidly neutralize the acid in the liquid precursor at the interface, forcibly raising the pH of the microenvironment to the physiological range (approximately 7.4). This pH increase is the decisive condition for triggering in-situ gelation. The mechanism is as follows:

[0014] The increase in pH value leads to the deprotonation of the carboxyl groups of anionic polysaccharides and the phenolic hydroxyl groups of plant polyphenols, resulting in a significant increase in negative charge density, which greatly enhances the electrostatic attraction between the positive charge carried by cationic polymers.

[0015] At the same time, the deprotonation of functional groups also activates and strengthens the coordination crosslinking between divalent cations and polymer chains;

[0016] The two forces mentioned above promote the formation of a suitable conformation of the polymer chain, thereby consolidating the hydrogen bond network with plant polyphenols at its core.

[0017] These three non-covalent forces, triggered by an increase in pH, occur synergistically and rapidly, jointly constructing a stable three-dimensional network that allows the liquid precursor to transform into a solid gel within minutes. This transformation process begins at the bottom of the wound bed and solidifies layer by layer from bottom to top, ultimately forming a homogeneous gel layer that perfectly conforms to the wound contour. The resulting gel possesses complex functions due to the inherent properties of its components: the cationic polymer, plant polyphenols, and divalent cation source work together to achieve broad-spectrum bactericidal effects through multiple pathways, including disrupting bacterial cell membrane structures and inhibiting key enzyme activity; simultaneously, the cationic polymer can aggregate negatively charged blood cells, and the plant polyphenols can constrict blood vessels and activate coagulation pathways, thereby achieving hemostasis.

[0018] Preferably, in step S3, the in-situ gelation is triggered by the pH value of the liquid precursor rising to the physiological pH range.

[0019] By adopting the above technical solution, it was clarified that the gelation mechanism of the system is a pH-responsive mechanism, and its phase transition is controlled by specific external pH environment changes, rather than time-dependent or temperature-dependent gelation.

[0020] Preferably, the cationic polymer in step S1 is selected from at least one of chitosan or ε-polylysine.

[0021] By adopting the above technical solutions, both chitosan and ε-polylysine have definite biocompatibility and inherent antibacterial activity. The amino groups on their molecular chains are protonated in aqueous solution and carry a positive charge, which provides a basis for the pH-responsive electrostatic attraction.

[0022] Preferably, the anionic polysaccharide in step S2 is sodium alginate.

[0023] By adopting the above technical solution, the carboxyl group on the sodium alginate molecular chain is the key functional group that coordinates and crosslinks with divalent cations, and is also the main source of generating negative charges to carry out electrostatic attraction after pH increases.

[0024] Preferably, the plant polyphenol in step S2 is tannic acid.

[0025] By adopting the above technical solution, the tannic acid molecule contains a large number of phenolic hydroxyl groups, which enable it to serve as the core of the hydrogen bond network and provide a negative charge after the pH increases. At the same time, it can effectively interact with proteins to achieve astringent hemostatic and antibacterial functions.

[0026] Preferably, the divalent cation source in step S2 is selected from at least one of zinc gluconate or calcium chloride.

[0027] By employing the above technical solution, zinc gluconate or calcium chloride can dissociate into Zn in aqueous solution.2+ or Ca 2+ Zn acts as an ionic crosslinking point to connect polymer chains. 2+ It also has the function of inhibiting bacterial growth, which can enhance the overall bactericidal performance of the system.

[0028] Preferably, in solution A, the mass-volume concentration of the cationic polymer is 1.5% to 3.0%; in solution B, the mass-volume concentration of the anionic polysaccharide is 0.5% to 2.5%, the mass-volume concentration of the plant polyphenol is 0.5% to 2.5%, and the mass-volume concentration of the divalent cation source is 0.2% to 1.5%.

[0029] By adopting the above technical solution, the above concentration range can ensure that the mixed system has a suitable crosslinking point density, thereby ensuring that the gel has sufficient mechanical strength for use as a dressing while obtaining a gelation time within a specific range.

[0030] Preferably, the preparation method further includes the following steps:

[0031] S4. Prepare an unlocking solution, wherein the unlocking solution is an aqueous solution containing a chelating agent;

[0032] The unlocking fluid is used to contact the medical dressing when needed, so as to change the medical dressing from a gel state to a liquid state, thereby enabling its removal.

[0033] By adopting the above technical solution, a controllable dressing removal mechanism is provided. Since the gel network is maintained by non-covalent bonds, specific chemical components in the unlocking solution can disrupt these non-covalent forces, causing the network structure to disintegrate, thus achieving non-destructive, on-demand removal of the dressing.

[0034] Preferably, the chelating agent in the unlocking solution is selected from at least one of citrate or ethylenediaminetetraacetic acid.

[0035] By employing the above-mentioned technical solution, the citrate or ethylenediaminetetraacetic acid anions exhibit a higher affinity for divalent cations than the functional groups on the polymer chain. When the unlocking solution comes into contact with the gel, the chelating agent competitively binds to the divalent cations in the gel network, thereby disrupting the coordination crosslinking of metal ions. This is the main mechanism of gel disintegration.

[0036] Preferably, the pH value of the unlocking solution is 5.5 to 6.5.

[0037] By adopting the above technical solution, this pH range helps the chelating agent to exert its chelating function and can simultaneously change the protonation state of the functional groups on the polymer chain, weaken the electrostatic attraction and hydrogen bonding between chains, and synergistically promote the rapid and complete disintegration of the gel network.

[0038] In summary, the present invention has at least one of the following beneficial technical effects:

[0039] 1. The preparation method of the present invention ensures that the pH of solution B, containing anionic polysaccharides, plant polyphenols, and a divalent cation source, is pre-adjusted to an acidic range of 4.5–6.0, guaranteeing good fluidity of the liquid precursor formed after mixing with solution A before application. Upon contact with a physiological pH environment, this liquid precursor can trigger rapid in-situ gelation due to the increased pH value. This technical feature allows the dressing to closely conform to irregular wound contours and form a continuous, uniform physical barrier on the wound surface, avoiding the problems of poor adhesion or incomplete coverage caused by shape mismatch in traditional pre-formed dressings.

[0040] 2. This invention achieves a dual function of bactericidal and hemostatic properties in the final dressing by specifically combining cationic polymers, plant polyphenols, and divalent cationic sources. The cationic polymers aggregate blood cells through electrostatic interactions, while the plant polyphenols promote coagulation through astringent effects and activation of coagulation pathways. Simultaneously, the divalent cationic source (such as zinc gluconate) works in conjunction with the other two to inhibit bacterial activity through multiple pathways. The synergistic effect of these components allows the dressing to provide comprehensive hemostasis and infection prevention while simultaneously sealing the wound.

[0041] 3. The dressing prepared in this invention has a three-dimensional network structure based on reversible non-covalent bonds (including metal ion coordination). This allows the dressing to undergo structural disintegration upon contact with an unlocking solution containing a specific chelating agent, transforming from a solid gel into a liquid. This on-demand liquefaction removal method avoids the mechanical tearing and secondary damage to newly formed granulation tissue or healing epithelium caused by traditional dressings during replacement, achieving non-destructive removal of the dressing. Detailed Implementation

[0042] The main raw materials and reagents used in the following examples and comparative examples have the following sources and specifications. Reagents not specifically mentioned are all commercially available analytical grade or higher grade products.

[0043] Chitosan, CAS No.: 9012-76-4, is medical grade. The chitosan used in this embodiment of the invention has a degree of deacetylation of 92.5% and a weight-average molecular weight of 2.2 × 10⁻⁶, as determined by gel permeation chromatography. 5 Da.

[0044] ε-Polylysine, CAS No.: 28211-04-3, is medical grade. The ε-Polylysine used in the embodiments of this invention has a weight-average molecular weight of 4000 Da.

[0045] Sodium alginate, CAS No.: 9005-38-3, is medical grade. It is a block copolymer composed of β-D-mannuronic acid and α-L-guluronic acid linked by (1→4) bonds. The sodium alginate used in the embodiments of this invention has a guluronic acid / mannuronic acid ratio of 1.2 and a weight-average molecular weight of 1.8 × 10⁻⁶. 5 Da.

[0046] Staphylococcus aureus, strain preservation number ATCC 6538.

[0047] Escherichia coli, strain preservation number ATCC 8739.

[0048] Fresh anticoagulated rabbit blood was obtained from healthy New Zealand white rabbits, using a 3.8% (w / v) trisodium citrate solution as the anticoagulant, with a blood-to-anticoagulant volume ratio of 9:1.

[0049] Example 1: This example provides a method for preparing and applying a medical dressing, the steps of which are as follows:

[0050] 1. Preparation of solution A:

[0051] Weigh out 2.0 g of chitosan (degree of deacetylation 92.5%, Mw = 2.2 × 10⁻⁶). 5 Chitosan powder (Da). Separately, take 100 mL of water for injection and add 0.5 mL of glacial acetic acid, mixing thoroughly to obtain a 0.5% (v / v) acetic acid aqueous solution. At room temperature (25°C), slowly add the weighed chitosan powder to the above acetic acid aqueous solution with magnetic stirring (400 rpm). Continue stirring for 8 hours until the powder is completely dissolved, forming a homogeneous, clear, viscous solution free of insoluble matter. After terminal irradiation sterilization, aseptically aliquot the solution into containers, seal, and store for later use to obtain solution A.

[0052] 2. Preparation of solution B:

[0053] Weigh out 1.5 g of sodium alginate, 1.5 g of tannic acid, and 1.0 g of zinc gluconate. At room temperature (25°C), take 100 mL of water for injection and place it on a magnetic stirrer (400 rpm). First, add the sodium alginate and stir until completely dissolved. Then, slowly add the tannic acid and zinc gluconate sequentially, continuing to stir until all components are completely dissolved, forming a homogeneous, brownish-yellow, transparent solution. Using 0.1 M sodium hydroxide solution, add dropwise while monitoring the pH value in real time, precisely adjusting the pH of the mixed solution to 5.5. Filter the pH-adjusted solution through a sterile 0.22 μm pore size filter membrane for sterilization, aseptically dispense into light-proof containers, seal, and store for later use, obtaining solution B.

[0054] 3. Preparation of unlocking solution:

[0055] Weigh 5.0 g of trisodium citrate dihydrate and dissolve it in 100 mL of physiological saline, stirring until completely dissolved. Adjust the pH of the solution to 6.0 using 0.1 M citric acid solution. Filter the prepared solution through a sterile 0.22 μm pore size filter membrane for sterilization, aseptically dispense into containers, and seal for later use.

[0056] 4. Application and Removal Methods:

[0057] In use, draw equal volumes of solution A and solution B using a dual-channel sterile syringe, and simultaneously squeeze them into a static mixer to quickly and evenly mix them. Apply the mixed liquid precursor immediately to the target wound. After application, the liquid precursor gels in situ at the wound site. When dressing removal is required, take sterile gauze, thoroughly soak it in the prepared unlocking solution, and apply it wet to the surface of the gel dressing, leaving it in place for 10 minutes. Afterward, rinse the wound with sterile saline to completely remove any liquefied dressing residue.

[0058] Example 2: This example provides a method for preparing and applying a medical dressing, the steps of which are as follows:

[0059] 1. Preparation of solution A:

[0060] Weigh 2.5 g of ε-polylysine (Mw = 4000 Da). At room temperature (25°C), slowly add it to 100 mL of water for injection while magnetically stirring (400 rpm). Continue stirring for 2 hours until completely dissolved, forming a homogeneous and clear solution. After terminal irradiation sterilization, aseptically aliquot the solution into containers, seal, and store for later use to obtain solution A.

[0061] 2. The preparation of solution B, the preparation of the unlocking solution, and the methods for application and removal are the same as in Example 1.

[0062] Example 3: This example provides a method for preparing and applying a medical dressing, the steps of which are as follows:

[0063] 1. Preparation of solution B:

[0064] Weigh out 1.5 g of sodium alginate, 1.5 g of tannic acid, and 0.5 g of anhydrous calcium chloride. The preparation method is the same as step 2 of Example 1, except that zinc gluconate is replaced with calcium chloride.

[0065] 2. The preparation of solution A, the preparation of unlocking solution, and the methods of application and removal are the same as in Example 1.

[0066] Example 4: This example provides a method for preparing and applying a medical dressing, the steps of which are as follows:

[0067] 1. Preparation of solution A:

[0068] Weigh 1.5 g of chitosan and dissolve it in 100 mL of 1.0% (v / v) aqueous acetic acid solution. The remaining preparation steps are the same as step 1 in Example 1.

[0069] 2. Preparation of solution B:

[0070] Weigh 0.5 g of sodium alginate, 0.5 g of tannic acid, and 0.2 g of zinc gluconate, dissolve them in 100 mL of water for injection, and precisely adjust the pH of the final solution to 4.5. The remaining preparation steps are the same as step 2 in Example 1.

[0071] 3. The preparation, application, and removal methods of the unlocking fluid are the same as in Example 1.

[0072] Example 5: This example provides a method for preparing and applying a medical dressing, the steps of which are as follows:

[0073] 1. Preparation of solution A:

[0074] Weigh 3.0 g of chitosan and dissolve it in 100 mL of 0.25% (v / v) aqueous acetic acid solution. The remaining preparation steps are the same as step 1 in Example 1.

[0075] 2. Preparation of solution B:

[0076] Weigh 2.5 g of sodium alginate, 2.5 g of tannic acid, and 1.5 g of zinc gluconate, dissolve them in 100 mL of water for injection, and precisely adjust the pH of the final solution to 6.0. The remaining preparation steps are the same as step 2 in Example 1.

[0077] 3. The preparation, application, and removal methods of the unlocking fluid are the same as in Example 1.

[0078] Comparative Example 1:

[0079] Compared with Example 1, the difference is that no tannic acid is added during the preparation of solution B; that is, solution B contains only 1.5% (w / v) sodium alginate and 1.0% (w / v) zinc gluconate. The remaining components, proportions, and preparation methods are the same as in Example 1.

[0080] Comparative Example 2:

[0081] Compared with Example 1, the difference is that zinc gluconate is not added during the preparation of solution B; that is, solution B contains only 1.5% (w / v) sodium alginate and 1.5% (w / v) tannic acid. The remaining components, proportions, and preparation methods are the same as in Example 1.

[0082] Comparative Example 3:

[0083] The difference from Example 1 is that solution B contains only 1.5% (w / v) sodium alginate, without the addition of tannic acid or zinc gluconate. All other components, proportions, and preparation methods are the same as in Example 1.

[0084] Comparative Example 4:

[0085] This comparative example prepares a conventional, non-removable chemically cross-linked hydrogel. Specifically, solution A (2% chitosan solution) prepared in Example 1 is mixed with a 0.1% (w / v) glutaraldehyde aqueous solution at a volume ratio of 10:1, and reacted at 37°C for 4 hours to form a chemically cross-linked chitosan hydrogel. This comparative example does not involve the preparation of solution B or the unlocking solution.

[0086] Comparative Example 5:

[0087] The difference from Example 1 is that, in the dressing removal step, sterile saline solution was used instead of the unlocking solution prepared in Example 1 for wet dressing and rinsing. All other components, preparation methods, and application methods are the same as in Example 1.

[0088] Comparative Example 6:

[0089] Compared with Example 1, the difference is that in the preparation of the unlocking solution, disodium ethylenediaminetetraacetate (EDTA-2Na) is used instead of trisodium citrate. The other components, proportions and preparation methods of the unlocking solution, as well as the preparation methods of solution A and solution B and the application methods of the dressing are the same as in Example 1.

[0090] Test Example 1:

[0091] This test was used to determine the gelation time of the liquid precursors of Examples 1-5 and Comparative Examples 1-3 under simulated physiological conditions.

[0092] The experimental steps are as follows:

[0093] 1. Take several clean 10 ml vials and place them in a constant temperature water bath at 37°C for 10 minutes for preheating.

[0094] 2. Using two separate 1 ml sterile syringes, draw 1.0 ml of solution A and 1.0 ml of solution B to be tested, respectively.

[0095] 3. Quickly and synchronously inject the solutions from the two syringes into the preheated vials from step 1, and start the timer the instant the injection is completed.

[0096] 4. Immediately vortex the vial for 3 seconds to ensure that the two solutions are mixed evenly, and then place it in a constant temperature water bath at 37°C.

[0097] 5. Starting from the start of the timing, remove the vial from the water bath every 15 seconds, slowly tilt it to a 90-degree angle with the horizontal plane, hold it for 2 seconds, and observe the flow of the mixture inside the vial.

[0098] 6. Record the time from the start of the timer until the three-dimensional network structure formed by the mixture in the vial ceases macroscopic flow when the vial is inverted. This time point is defined as the gelation time.

[0099] 7. If the mixture fails to form a non-flowing gel within 1800 seconds (30 minutes), it is recorded as failing to form a stable gel within the specified time.

[0100] 8. For each embodiment and comparative example, repeat the above experimental steps three times, and take the arithmetic mean of the three measurement times as the final gelation time.

[0101] The test results are shown in Table 1:

[0102] Table 1. Results of gelation performance tests for Examples 1-5 and Comparative Examples 1-3:

[0103] Example / Comparative Example Number Gelation time (seconds) Example 1 183 Example 2 167 Example 3 212 Example 4 276 Example 5 148 Comparative Example 1 554 Comparative Example 2 Failed to form a stable gel within 1800 seconds Comparative Example 3 Failed to form a stable gel within 1800 seconds

[0104] The test results in Table 1 show that the two-component liquid precursors prepared in Examples 1 to 5 can rapidly form stable hydrogels under simulated physiological conditions, with gelation times ranging from 148 seconds to 276 seconds. In contrast, the gelation time of Comparative Example 1 is significantly prolonged, while Comparative Examples 2 and 3 failed to form stable gel structures within an observation time of 1800 seconds. This result demonstrates that the composition system described in this invention possesses the ability to rapidly complete the liquid-to-solid phase transition under specific conditions.

[0105] The aforementioned gelation process occurs based on the synergistic triggering of multiple non-covalent interactions within the system as the pH changes from an unmixed acidic state (pH 4.5–6.0) to a neutral state (approximately pH 7.4) after contact with the physiological environment. During this pH transition, the protonation degree of the amino groups on the chitosan or ε-polylysine molecular chains decreases, exhibiting enhanced positive charge; simultaneously, the carboxyl groups of sodium alginate and the phenolic hydroxyl groups of tannic acid are deprotonated, exhibiting negative charge. The appearance of these two opposite charges promotes the formation of strong electrostatic attraction between polymer chains.

[0106] Meanwhile, divalent cations (such as Zn) 2+ or Ca 2+The tannic acid molecules form coordinate crosslinks with the carboxyl, hydroxyl, and amino functional groups on the polymer chains. The numerous phenolic hydroxyl groups on the tannic acid molecules form extensive hydrogen bond networks with other polar groups on the polymer chains. It is this multi-layered dynamic crosslinking, composed of electrostatic attraction, metal ion coordination, and hydrogen bond networks, that works together to construct a dense three-dimensional network structure, thereby binding free-flowing water molecules within it, macroscopically manifesting as a rapid transformation from a liquid precursor to a solid gel. The results of Comparative Examples 1, 2, and 3 further confirm that without tannic acid, divalent cations, or both, the system cannot form a crosslinked network of sufficient strength and density, thus failing to achieve rapid and effective gelation.

[0107] Test Example 2:

[0108] This test was used to determine the structural dissociation properties of the hydrogel dressing prepared in Example 1 under the action of a specific unlocking solution.

[0109] The experimental steps are as follows:

[0110] 1. Take solutions A and B prepared in Example 1 and mix them in a sterile petri dish at a volume ratio of 1:1, with a total volume of 4 ml. Incubate the petri dish at 37°C for 30 minutes to form a hydrogel film of uniform thickness.

[0111] 2. Using a circular sampler with a diameter of 10 mm, prepare a disc-shaped gel sample from the gel membrane prepared in step 1.

[0112] 3. Gently absorb excess moisture from the surface of the prepared gel sample with filter paper, and then immediately place it on an analytical balance with an accuracy of 0.1 mg to weigh its initial wet weight and record it as W0.

[0113] 4. Completely immerse the weighed gel sample in 10 ml of the unlocking solution prepared in Example 1, which is placed in a sealed container.

[0114] 5. Place the container in a constant temperature shaker at 37°C and shake it horizontally at a rate of 50 rpm for 15 minutes.

[0115] 6. After processing, visually observe the morphology of the gel sample. If there are still solid residues, carefully remove it from the solution and gently rinse it three times with 10 ml of deionized water to remove any adhering unlocking solution.

[0116] 7. After rinsing, blot the surface moisture of the residue with filter paper, and weigh it again using an analytical balance. Record the final wet weight as Wf. If there is no solid residue, record Wf as 0.

[0117] 8. Calculate the gel dissolution rate using the following formula: Gel dissolution rate (%) = (W0 - Wf) / W0 × 100%.

[0118] 9. Repeat the above experimental steps three times, and take the arithmetic mean of the three measurements as the final gel dissolution rate.

[0119] The test results are shown in Table 2:

[0120] Table 2. Solubility test results of the hydrogel prepared in Example 1 in the unlocking solution:

[0121] Experiment number <![CDATA[Initial wet weight W0 (g)]]> Final wet weight Wf (grams) Gel dissolution rate (%) 1 0.152 0.005 96.71 2 0.149 0.007 95.3 3 0.155 0.004 97.42 average value - - 96.48

[0122] Table 2 shows that the hydrogel sample prepared according to Example 1 achieved an average gel dissolution rate of 96.48% after 15 minutes of contact with a specific unlocking solution. This data indicates that the original solid gel network structure underwent significant disintegration under the action of the unlocking solution, transforming into a state that can be removed by fluid rinsing. This result confirms that the hydrogel system possesses the ability to controllably transition from a solid to a liquid state under specific chemical conditions.

[0123] The mechanism of this solid-liquid phase transition is rooted in the competitive interaction between specific components in the unlocking solution and the multiple dynamic cross-linking sites constituting the gel network. The citrate anions in the unlocking solution have a competitive effect on the divalent zinc ions (Zn) in the system. 2+ Zn exhibits stronger chelating ability than functional groups on polymer chains (such as carboxyl and hydroxyl groups). This preferential chelation leads to Zn... 2+ The metal ion coordination crosslinking is extracted from the original coordination crosslinking sites that maintain the network structure, thereby causing the metal ion coordination crosslinking to fail.

[0124] Meanwhile, the weakly acidic environment (pH 5.0–6.5) of the unlocking solution altered the protonation state of the acid and basic groups on the polymer chains in the gel network. This weakened the electrostatic attraction between the amino groups on the chitosan chains and the negatively charged groups on sodium alginate and tannic acid, and simultaneously disrupted the hydrogen bond network formed around the tannic acid core. The simultaneous breakage of these non-covalent forces (metal ion coordination, electrostatic attraction, and hydrogen bond network) led to the collapse of the three-dimensional network structure that maintained the gel's solid state. Ultimately, the polymer chains, freed from cross-linking constraints, redispersed in the aqueous phase, restoring the entire system from a solid gel to a low-viscosity liquid state, thus enabling removal without the application of mechanical force.

[0125] Test Example 3:

[0126] This test was used to compare the promoting effects of the samples from Examples 1-5 and Comparative Examples 1-3 on blood coagulation through an in vitro whole blood coagulation experiment.

[0127] The experimental steps are as follows:

[0128] 1. The liquid precursors of Examples 1-5 and Comparative Examples 1-3 were prepared into gels under sterile conditions, and 100.0 mg of gel sample was weighed and placed in a clean 1.5 mL centrifuge tube.

[0129] 2. Set up a negative control group by adding 100.0 mg of sterile saline to the centrifuge tubes of this group. Also set up a blank control group by adding no sample to the centrifuge tubes of this group.

[0130] 3. Preheat all centrifuge tubes in a 37°C water bath for 5 minutes.

[0131] 4. Use a pipette to add 200 μL of fresh anticoagulated rabbit blood to each centrifuge tube (including the negative control group and the blank control group), and start the timer immediately.

[0132] 5. Incubate all centrifuge tubes at 37°C for another 10 minutes without shaking.

[0133] 6. Immediately after incubation, add 1.0 ml of water for injection to each centrifuge tube to lyse the free red blood cells that did not participate in the formation of blood clots and release hemoglobin.

[0134] 7. Centrifuge all centrifuge tubes at 4000 rpm for 5 minutes to allow blood clots and cell debris to precipitate.

[0135] 8. Carefully aspirate the supernatant from each tube and measure its absorbance (OD value) at 540 nm using a UV-Vis spectrophotometer. The absorbance value of the blank control group is recorded as OD blank, the absorbance value of the negative control group is recorded as OD negative, and the absorbance value of each sample group is recorded as OD sample.

[0136] 9. Calculate the blood coagulation index (BCI) of each sample using the following formula: Blood coagulation index (%) = [(OD sample - OD blank) / (OD negative - OD blank)] × 100%. The lower the blood coagulation index, the stronger the effect of the sample in promoting blood coagulation.

[0137] 10. For each embodiment and comparative example, repeat the above experimental steps three times, and take the arithmetic mean of the three measurement results as the final blood coagulation index.

[0138] The test results are shown in Table 3:

[0139] Table 3. Results of in vitro blood coagulation index tests for Examples 1-5 and Comparative Examples 1-3:

[0140] Example / Comparative Example Number Blood coagulation index (BCI, %) Example 1 24.8 Example 2 28.3 Example 3 25.9 Example 4 33.1 Example 5 22.7 Comparative Example 1 51.2 Comparative Example 2 43.6 Comparative Example 3 78.9

[0141] The test results in Table 3 show that the blood coagulation indices (BCIs) of Examples 1 to 5 were significantly lower than those of Comparative Examples 1, 2, and 3. Specifically, the BCI values ​​of Examples 1 to 5 ranged from 22.7% to 33.1%, while the BCI values ​​of Comparative Examples 1, 2, and 3 ranged from 43.6% to 78.9%. This data indicates that the composition system of the present invention, comprising a cationic polymer, anionic polysaccharide, plant polyphenols, and a divalent cationic source, can more effectively promote blood coagulation compared to systems lacking plant polyphenols or divalent cationic sources, and systems containing only cationic polymers and anionic polysaccharides.

[0142] The system's ability to promote blood clotting stems from the synergistic effect of its multiple components at the physical and chemical levels. First, the positive charge of the cationic polymers (such as chitosan) in the system allows for electrostatic attraction to the negatively charged surfaces of red blood cells and platelets, causing them to aggregate on the gel surface and providing an initial physical concentration effect for clot formation. Simultaneously, the three-dimensional network structure formed by the gel itself provides a physical barrier, absorbing wound exudate and concentrating clotting factors.

[0143] Furthermore, the tannic acid component in the system, with its numerous phenolic hydroxyl groups, can form hydrogen bonds and hydrophobic interactions with proteins in the blood (including coagulation factors), leading to conformational changes in the proteins and thus activating the intrinsic coagulation pathway. In addition, the astringent effect of tannic acid can constrict capillaries around the wound, reducing bleeding. The comparison results between the examples and Comparative Example 1 confirm the role of tannic acid in enhancing hemostasis. The comparison results between the examples and Comparative Examples 2 and 3 indicate that the divalent cation source and the complete combination of all components are necessary to achieve optimal procoagulant function.

[0144] Test Example 4:

[0145] This test was used to quantitatively compare the bactericidal effects of the samples from Examples 1-5 and Comparative Examples 1-3 on Staphylococcus aureus and Escherichia coli using the bacterial suspension contact method.

[0146] The experimental steps are as follows:

[0147] 1. The liquid precursors of Examples 1-5 and Comparative Examples 1-3 were prepared into gels in a sterile operating table, and 100.0 mg of gel sample was weighed and placed in a sterile 1.5 mL centrifuge tube.

[0148] 2. Staphylococcus aureus and Escherichia coli were inoculated separately into liquid culture medium and cultured at 37°C and 150 rpm with shaking until the logarithmic growth phase. The bacterial concentration was adjusted to approximately 1 × 10⁻⁶ using sterile phosphate-buffered saline (PBS, pH 7.4). 6 CFU / mL.

[0149] 3. Set up a blank control group, in which no gel sample is added to the centrifuge tubes.

[0150] 4. Add 1.0 mL of the bacterial suspension prepared in step 2 to all centrifuge tubes (including the sample group and the blank control group).

[0151] 5. Place all centrifuge tubes in a constant temperature shaker at 37°C and incubate at 100 rpm for 4 hours.

[0152] 6. After incubation, aspirate the bacterial culture from each centrifuge tube and perform 10-fold serial dilutions using sterile PBS.

[0153] 7. Take 100 μL of bacterial solution with an appropriate dilution and spread it evenly on a nutrient agar plate.

[0154] 8. Place all plates in a constant temperature incubator at 37°C and incubate for 24 hours, then count the colonies on the plates (CFU).

[0155] 9. Calculate the sterilization rate of each sample according to the following formula: Sterilization rate (%) = [(CFU blank control - CFU sample) / CFU blank control] × 100%.

[0156] 10. For each example and comparative example, as well as for the two test strains, the above experimental steps were repeated three times, and the arithmetic mean of the three measurements was taken as the final sterilization rate.

[0157] The test results are shown in Table 4:

[0158] Table 4. Results of in vitro bactericidal rate tests on the two strains in Examples 1-5 and Comparative Examples 1-3:

[0159] Example / Comparative Example Number Sterilization rate against Staphylococcus aureus (%) Sterilization rate against Escherichia coli (%) Example 1 99.7 99.5 Example 2 98.9 98.6 Example 3 99.8 99.4 Example 4 97.5 96.9 Example 5 99.9 99.85 Comparative Example 1 91.3 88.7 Comparative Example 2 94.6 92.1 Comparative Example 3 68.2 61.5

[0160] Table 4 shows that the compositions of Examples 1 to 5 exhibited high bactericidal rates against both Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, with values ​​exceeding 96%. In contrast, the bactericidal rates of Comparative Examples 1, 2, and 3 were significantly lower, especially Comparative Example 3, which contained only cationic polymers and anionic polysaccharides, with a bactericidal rate below 70%. This data indicates that the complete composition system of the present invention possesses broad-spectrum and highly efficient antibacterial properties.

[0161] The antibacterial function is achieved through the synergistic effect of multiple active components in the system. First, the positive charge of the cationic polymer chains (such as chitosan or ε-polylysine) in the system can electrostatically adsorb onto the negatively charged phospholipids and lipopolysaccharides on the bacterial cell membrane surface, disrupting the integrity of the membrane structure and leading to leakage of cell contents. Second, plant polyphenolic components (such as tannic acid) can interact with proteins on the bacterial cell wall and cell membrane, inhibiting the activity of key enzymes and interfering with the normal metabolic processes of bacteria.

[0162] In addition, divalent zinc ions (Zn) are present in the system. 2+ Tannin itself is an antibacterial agent. It can inactivate various enzymes in bacteria by binding to sulfhydryl groups in proteins, thereby inhibiting bacterial growth and reproduction. The combination of these three different mechanisms of action constitutes a multi-faceted attack mechanism against bacteria. The test results of Comparative Examples 1, 2, and 3 further confirm this: the lack of tannic acid (Comparative Example 1) or zinc ions (Comparative Example 2) both lead to a decrease in antibacterial performance, while the simultaneous lack of both components (Comparative Example 3) results in a significant reduction in antibacterial performance. This indicates that each component is necessary to achieve a high bactericidal rate.

[0163] Test Example 5:

[0164] This test is used to quantitatively compare the removability of the gels prepared in the embodiments of the present invention and conventional chemically cross-linked gels, and to verify the dependence of the removal function on specific unlocking solution components.

[0165] The experimental steps are as follows:

[0166] 1. Hydrogels were prepared according to the methods of Example 1, Example 3 and Comparative Example 4, respectively.

[0167] 2. Following steps 2 to 3 of test 1.2, prepare the above gel into disc-shaped samples with a known initial wet weight (W0).

[0168] 3. Set up the following five experimental groups:

[0169] Group A: The gel sample prepared in Example 1 was immersed in the unlocking solution (containing sodium citrate) prepared in Example 1.

[0170] Group B: The gel sample prepared in Example 1 was immersed in sterile physiological saline (simulating Comparative Example 5).

[0171] Group C: The chemically cross-linked gel sample prepared in Comparative Example 4 was immersed in the unlocking solution prepared in Example 1.

[0172] Group D: The gel sample prepared in Example 3 (using Ca) 2+ (Cross-linked), soaked in the unlocking solution prepared in Example 1.

[0173] Group E: The gel sample prepared in Example 1 was immersed in the unlocking solution (containing EDTA-2Na) prepared in Comparative Example 6.

[0174] 4. Place each group of samples in a constant temperature shaker at 37℃ and process at a rate of 50 rpm for 15 minutes.

[0175] 5. After the treatment is completed, follow steps 6 to 8 of Test Example 2 to measure the final wet weight (Wf) of each group of samples and calculate the gel dissolution rate.

[0176] 6. For each experimental group, repeat the above experimental steps three times, and take the arithmetic mean of the three measurements as the final gel dissolution rate.

[0177] The test results are shown in Table 5:

[0178] Table 5. Performance comparison test results of on-demand removal between the examples and the comparative examples:

[0179] experimental group Gel sample source Treatment solution Average gel dissolution rate (%) Group A Example 1 Example 1 Unlocking Liquid 97.1 Group B Example 1 physiological saline 2.8 Group C Comparative Example 4 Example 1 Unlocking Liquid 1.5 Group D Example 3 Example 1 Unlocking Liquid 95.8 Group E Example 1 Comparative Example 6 Unlocking Fluid 98.2

[0180] Table 5 shows that the gel samples prepared in Examples 1 and 3 exhibited high average gel dissolution rates, ranging from 95.8% to 98.2%, after contact with unlocking solutions containing specific chelating agents (groups A, D, and E). In contrast, the average gel dissolution rates of the gel sample from Example 1 in physiological saline (group B) and the chemically cross-linked gel from Comparative Example 4 in unlocking solution (group C) were both below 3.0%. This data confirms that the removability of the gel system described in this invention depends on the chemical action of the specific unlocking solution, and that this action is ineffective for conventional chemically cross-linked gels.

[0181] The high gel dissolution rates observed in groups A, D, and E are due to the chelating agents (citrate or EDTA) in the unlocking solution disrupting the three-dimensional network structure of the gel. These chelating agents interact with divalent metal cations (Zn) in the network. 2+ or Ca 2+ The polymer binds with high affinity, competitively replacing the original coordination bonds between the polymer functional groups and the metal ions, leading to the failure of the metal ion coordination crosslinking sites. Simultaneously, the specific pH environment of the unlocking solution alters the protonation state of the functional groups on the polymer chain, thereby weakening the electrostatic attraction and hydrogen bonding between chains.

[0182] The simultaneous disintegration of multiple dynamic non-covalent cross-linked networks causes the polymer chains constituting the gel backbone to lose their binding and redisperse in the aqueous medium, macroscopically manifesting as the liquefaction of the gel solid. The low solubility results in group B indicate that the gel network is stable in physiological saline without a specific chemical trigger (chelating agent). The results in group C show that the mechanism of action of this unlocking solution is ineffective against stable covalent bonds (such as CN bonds formed by glutaraldehyde cross-linking), thus clarifying that the on-demand removal function described in this invention is a unique property based on the reversibility of dynamic chemical bonds.

[0183] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method of preparing a bactericidal hemostatic medical dressing, characterized in that, The method comprises the following steps: S1, preparing solution A, which is an aqueous solution containing cationic polymer; S2, preparing solution B, which comprises dissolving anionic polysaccharide, plant polyphenol and divalent cation source in aqueous solution, and adjusting the pH value of the solution B to 4.5-6.0; S3, mixing the solution A with the solution B to form a liquid precursor capable of in-situ gelation after contacting physiological pH environment, thereby preparing the medical dressing; The plant polyphenol is tannic acid; The divalent cation source in the step S2 is selected from at least one of zinc gluconate or calcium chloride; In the step S3, the in-situ gelation is triggered by the increase of the pH value of the liquid precursor to the physiological pH range; The cationic polymer in the step S1 is selected from at least one of chitosan or ε-polylysine; The anionic polysaccharide in the step S2 is sodium alginate.

2. The method of preparing a bactericidal hemostatic medical dressing according to claim 1, characterized in that, In the solution A, the mass-volume concentration of the cationic polymer is 1.5%-3.0%; In the solution B, the mass-volume concentration of the anionic polysaccharide is 0.5%-2.5%, the mass-volume concentration of the plant polyphenol is 0.5%-2.5%, and the mass-volume concentration of the divalent cation source is 0.2%-1.5%.

3. The method of preparing a bactericidal hemostatic medical dressing according to claim 1 or 2, characterized in that, The method further comprises the following step: S4, preparing unlocking liquid, which is an aqueous solution containing chelating agent; The unlocking liquid is used to contact the medical dressing when necessary, so as to make the medical dressing change from gel state to liquid state, thereby achieving removal.

4. The method of preparing a bactericidal hemostatic medical dressing according to claim 3, characterized in that, The chelating agent in the unlocking liquid is selected from at least one of citrate or ethylenediaminetetraacetate.

5. The method of preparing a bactericidal hemostatic medical dressing according to claim 3, characterized in that, The pH value of the unlocking liquid is 5.5-6.5.