Soluble hemostatic amphotericin B grafted cotton yarn dressing and preparation method thereof

Abstract

The invention discloses a soluble hemostatic amphotericin B grafted cotton yarn dressing and a preparation method thereof, and relates to the technical field of biomedical materials. According to the soluble hemostatic amphotericin B grafted cotton yarn dressing, soluble cotton yarn with the etherification degree being 0.3-0.5 serves as a substrate, and a double-response medicine grafting layer and a functional coating are sequentially constructed on the soluble cotton yarn. The method comprises the following steps: performing citric acid crosslinking and plasma activation on cotton yarns; covalently grafting the synthesized dual-response intelligent linker on the activated cotton yarn to obtain a dual-response drug grafting layer; finally, a quaternized chitosan / catechol modified hyaluronic acid nanofiber coating is deposited on the surface through electrostatic spinning, and the functional coating is obtained. The soluble hemostatic amphotericin B grafted cotton yarn dressing prepared by the invention has the advantages of rapid hemostasis, intelligent controlled drug release and good biocompatibility and degradability, and is especially suitable for treatment of complex infected wounds.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a soluble hemostatic amphotericin B grafted cotton gauze dressing and its preparation method. Background Technology

[0002] In clinical medicine, the management of deep wounds, burns, and surgical incisions faces the dual challenges of hemostasis and infection control. Traditional dressings, such as cotton gauze and bandages, primarily serve the functions of physical coverage and absorption of exudate, offering limited effectiveness for wounds with persistent bleeding or fungal infections. Existing functional dressings, such as medicated dressings, often employ simple physical mixing or embedding of drugs, resulting in problems such as burst drug release, short duration of action, lack of targeted delivery, and susceptibility to drug resistance. Particularly for highly effective but somewhat toxic antifungal drugs like amphotericin B, achieving precise and controlled release at the site of infection remains a technical challenge. Furthermore, an ideal dressing that combines rapid hemostasis, biodegradability, good biocompatibility, and controlled drug release remains a key research focus and challenge. Therefore, developing a composite dressing that intelligently responds to the wound microenvironment, achieves on-demand drug release, and possesses excellent hemostatic properties and biosafety has significant clinical implications and application value. Summary of the Invention

[0003] The purpose of this invention is to solve the problems mentioned in the background art and to provide a soluble hemostatic amphotericin B grafted cotton gauze dressing and its preparation method.

[0004] In a first aspect, the present invention provides a soluble hemostatic amphotericin B grafted cotton gauze dressing, the dressing having a multilayer structure including a base layer, a dual-response drug grafting layer, and a functional coating.

[0005] The degree of etherification of the base layer is 0.3-0.5.

[0006] A second aspect of the present invention provides a method for preparing a soluble hemostatic amphotericin B-grafted cotton gauze dressing, comprising the following steps:

[0007] Step 1: Place a 10×10cm soluble hemostatic cotton gauze sheet in anhydrous ethanol and deionized water for ultrasonic cleaning for 30 minutes each. After drying, immerse the cleaned cotton gauze in citric acid aqueous solution, remove and squeeze dry, crosslink in a 120℃ oven for 10 minutes, cool, wash with deionized water until neutral, dry, and treat with low-temperature oxygen plasma for 5 minutes to obtain activated cotton gauze, i.e., the base layer.

[0008] As a preferred embodiment of the present invention, in step one, the rolling drying operation needs to be rolled dry to a 100% liquid content;

[0009] As a preferred technical solution of the present invention, in step one, the ratio of the amount of washed cotton yarn to citric acid aqueous solution is 1g:(20-30)mL;

[0010] As a preferred embodiment of the present invention, in step one, the citric acid aqueous solution has a mass fraction of 0.5%;

[0011] As a preferred technical solution of the present invention, in step one, the power of the low-temperature oxygen plasma treatment is 100W;

[0012] It should be noted that step one, through citric acid crosslinking and plasma activation treatment of the cotton yarn substrate, fundamentally improves the core physical and chemical properties of the dressing. Citric acid crosslinking effectively enhances the stability of the cotton yarn fiber network, enabling it to maintain structural integrity in the moist wound environment, preventing premature dissolution and collapse, and providing a reliable carrier for long-term drug release. The subsequent plasma treatment efficiently introduces abundant active carboxyl groups onto the fiber surface. These groups act as anchor points, laying a solid foundation for the subsequent strong covalent grafting of amphotericin B via a smart linker, a prerequisite for constructing a stable and efficient drug delivery system.

[0013] Step 2: Immerse the activated cotton yarn obtained in Step 1 in MES buffer and activate it by shaking at room temperature for 30 min. Take out the cotton yarn and immerse it in PBS buffer containing a dual-response linker at pH 7.4. Add polyethylene glycol and Tween-80 as dispersants. Place the system in a constant temperature shaker at 45℃ and react at 100 rpm for 6 h. Wash 3-4 times with 90% ethanol solution and vacuum dry at 45℃. The dual-response drug graft layer is prepared to obtain AmB grafted cotton yarn.

[0014] As a preferred technical solution of the present invention, in step two, the ratio of activated cotton yarn, MES buffer, dual-response linker, buffer, polyethylene glycol, Tween-80 and 90% ethanol solution is 1g:(40-60)mL:0.05g:100mL:5mL:1mL:(500-1000)mL.

[0015] As a preferred embodiment of the present invention, in step two, the MES buffer is an MES buffer containing 0.1 mol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 0.05 mol / L N-hydroxysuccinimide (NHS) with a pH of 6.

[0016] As a preferred embodiment of the present invention, in step two, the molecular weight of the polyethylene glycol is 1200-1800;

[0017] As a preferred embodiment of the present invention, the preparation steps of the dual-response linker in step two are as follows:

[0018] S1. Dissolve amino-polyethylene glycol-carboxyl in dimethylformamide, add tert-butoxycarbonyl hydrazine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, react at room temperature for 12 h, add trifluoroacetic acid, stir at room temperature for 2-3 h to obtain hydrazine-terminated polyethylene glycol, react the obtained hydrazine-terminated polyethylene glycol with N-succinimide ester in an acetate-sodium acetate buffer solution at pH 5 to obtain polyethylene glycol-hydrazone-succinimide ester;

[0019] As a preferred embodiment of the present invention, the molecular weight of the amino-polyethylene glycol-carboxyl group is 2000;

[0020] As a preferred embodiment of the present invention, the ratio of amino-polyethylene glycol-carboxyl, tert-butoxycarbonylhydrazine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and trifluoroacetic acid is 2 mmol:20 mL:5 mmol:2.4 mmol:3 mmol:10 mL.

[0021] As a preferred embodiment of the present invention, the molar ratio of hydrazine-terminated polyethylene glycol to N-hydroxysuccinimide ester is 1:1.2, and the volume ratio of hydrazine-terminated polyethylene glycol to acetate-sodium acetate buffer is 1 mmol:20 mL.

[0022] As a preferred embodiment of the present invention, the concentration of the acetate-sodium acetate buffer solution is 0.1 mol / L.

[0023] S2. Weigh out the matrix metalloproteinase-sensitive peptide and dissolve it in phosphate buffer at pH 7.4. Add N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester and react at room temperature in the dark to obtain maleimide-terminated peptide.

[0024] As a preferred embodiment of the present invention, the matrix metalloproteinase-sensitive peptide is one of MMP-2 / 9 sensitive peptide, MMP-1 / 13 sensitive peptide, and MMP-3 / 7 sensitive peptide;

[0025] As a preferred embodiment of the present invention, the molar ratio of the matrix metalloproteinase-sensitive peptide and N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester is 1:1.2;

[0026] As a preferred embodiment of the present invention, the ratio of the matrix metalloproteinase-sensitive peptide to phosphate buffer is 1 mmol: 20 mL.

[0027] As a preferred embodiment of the present invention, the concentration of the phosphate buffer solution is 0.01 mol / L.

[0028] S3. Weigh out amphotericin B and 1,6-hexanediamine and dissolve them in dimethylformamide. Add 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide. React at room temperature in the dark to obtain amphotericin B-amino.

[0029] As a preferred embodiment of the present invention, the molar ratio of amphotericin B, 1,6-hexanediamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, and N-hydroxysuccinimide is 1:1.5:1.5:1.8.

[0030] As a preferred embodiment of the present invention, the ratio of amphotericin B to dimethylformamide is 1 mmol: 10 mL.

[0031] S4. The amphotericin B-amino obtained in S3 and the polyethylene glycol-hydrazone-succinimide ester obtained in S1 are dissolved in dimethylformamide and reacted. The resulting product is then dissolved in phosphate buffer at pH 7.4 and reacted in the dark to obtain a dual-response smart linker.

[0032] As a preferred embodiment of the present invention, the molar ratio of amphotericin B-amino to polyethylene glycol-hydrazone-succinimide ester is 1:1.05;

[0033] As a preferred embodiment of the present invention, the ratio of amphotericin B-amino to dimethylformamide is 1 mmol: 15 mL;

[0034] As a preferred embodiment of the present invention, the molar ratio of polyethylene glycol-hydrazone-succinimide ester to maleimide-terminated peptide is 1:1.05;

[0035] As a preferred embodiment of the present invention, the ratio of polyethylene glycol-hydrazone-succinimide ester to phosphate buffer is 1 mmol: 25 mL.

[0036] It should be noted that the preparation of the dual-response linker is the core of this intelligent drug release mechanism. Its design principle lies in the ingenious tandem linking of two response units: a pH-sensitive hydrazone bond and a matrix metalloproteinase (MMP)-sensitive peptide. Under normal physiological conditions, the linker remains stable; when the dressing is applied to an infected wound, the acidity of the wound microenvironment and the high expression of MMP enzymes trigger hydrazone bond hydrolysis and peptide chain breakage, respectively, thereby controlling the release rate and magnitude of amphotericin B. This dual-locking mechanism ensures efficient drug release only at the core of the infection, improving therapeutic targeting and reducing the risk of systemic toxicity, thus endowing the dressing with intelligent release properties.

[0037] It is important to note that the core function of step two is to firmly graft the dual-response linker molecule with intelligent responsive properties onto the activated cotton yarn substrate via stable covalent bonds. This immobilizes amphotericin B in a dormant state within the dressing, rather than through simple physical mixing, thus preventing initial drug release at the source. The resulting dual-response drug graft layer is a key structure for achieving long-lasting, controllable antifungal function in the dressing, ensuring drug stability during transportation and storage, and enabling on-demand activation during use.

[0038] Step 3: Prepare quaternized chitosan solution and catechol-modified hyaluronic acid aqueous solution respectively. Using biaxial electrospinning technology, deposit a functional coating on the surface of the AmB grafted cotton yarn obtained in Step 2. Set the feed rate to 0.8 mL / h, voltage to 18 kV, receiving distance to 15 cm, and continue for 30 min. Place the coated cotton yarn in PBS buffer at pH 7.4 and let it stand at 4℃ for 24 h to obtain crude dressing.

[0039] As a preferred technical solution of the present invention, in step three, the quaternized chitosan solution is prepared from quaternized chitosan, acetic acid and deionized water, wherein the mass fraction of quaternized chitosan is 2% and the mass fraction of acetic acid is 1%.

[0040] As a preferred technical solution of the present invention, in step three, the catechol-modified hyaluronic acid aqueous solution is prepared by catechol-modified hyaluronic acid and deionized water, wherein the mass fraction of catechol-modified hyaluronic acid is 1%.

[0041] As a preferred embodiment of the present invention, the preparation steps of the quaternized chitosan in step three are as follows:

[0042] Weigh 10g of chitosan and disperse it in 100mL of isopropanol. Slowly add 30mL of 2,3-epoxypropyltrimethylammonium chloride aqueous solution (50% by mass) while stirring. React at 70℃ for 12h. Precipitate with anhydrous ethanol, wash 3-4 times, and dry under vacuum at 45℃ to obtain quaternized chitosan.

[0043] As a preferred embodiment of the present invention, the degree of deacetylation of chitosan is ≥90%, and the molecular weight is 50kDa.

[0044] As a preferred embodiment of the present invention, the preparation steps of the catechin-modified hyaluronic acid in step three are as follows:

[0045] Sodium hyaluronate was weighed and dissolved in MES buffer at pH 5.0. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide were added and activated for 30 min. Dopamine hydrochloride was added and the mixture was stirred at room temperature in the dark for 24 h. The resulting reaction solution was dialyzed for 3 days using a dialysis bag with a molecular weight cutoff of 3500 and then freeze-dried to obtain catechol-modified hyaluronic acid.

[0046] As a preferred embodiment of the present invention, the sodium hyaluronate has a molecular weight of 200 kDa.

[0047] As a preferred embodiment of the present invention, the ratio of sodium hyaluronate, MES buffer, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride is 5g:500mL:2.5g:1.5g:3.0g.

[0048] It should be noted that in step three, a nanofiber functional coating composed of quaternized chitosan and catechol-modified hyaluronic acid was constructed on the outside of the drug-grafted layer using biaxial electrospinning technology. This coating not only endows the dressing with excellent flexibility and breathability, but more importantly, it achieves close adhesion to wound tissue and rapid hemostasis through the charge complementarity effect between components and the strong adhesive ability of catechol groups. In addition, the broad-spectrum antibacterial properties of quaternized chitosan itself synergistically work with the antifungal activity of amphotericin B, providing a three-dimensional protective barrier for the wound.

[0049] Step 4: Rinse the crude dressing obtained in Step 3 3-4 times with PBS buffer at pH 7.4, perform terminal sterilization using gamma rays, seal and package to obtain soluble hemostatic amphotericin B grafted cotton gauze dressing.

[0050] As a preferred embodiment of the present invention, in step four, the dose of the γ-ray is 25 kGy.

[0051] Compared with the prior art, the present invention has the following beneficial effects: (using a sub-system)

[0052] (1) By constructing a dual-response drug release mechanism of MMP enzyme and pH, this invention achieves precise and on-demand release of amphotericin B on infected wounds, effectively overcoming the shortcomings of uncontrollable drug release and easy burst release of traditional drug-loaded dressings, significantly improving the therapeutic effect and reducing toxic side effects.

[0053] (2) This invention modifies the cotton yarn substrate by crosslinking with citric acid and activating with plasma, and uses a covalent grafting strategy to fix the drug, so that the dressing maintains good biocompatibility and degradability while having excellent structural stability and drug loading stability, thus meeting the clinical needs for the mechanical properties and long-lasting effects of the dressing.

[0054] (3) This invention integrates rapid hemostasis function with intelligent antifungal function, and the functional layers work together to provide a multifunctional composite wound management solution, which is especially suitable for treating complex wounds that are susceptible to fungal infection.

[0055] In summary, the dressing provided by this invention combines intelligent drug release, rapid hemostasis, biodegradability, and good biocompatibility, effectively solving many pain points in the prior art and showing significant progress and broad clinical application prospects. Detailed Implementation

[0056] 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.

[0057] Preparation Example 1

[0058] The preparation steps of the dual-responsive linker are as follows:

[0059] S1. Dissolve 2 mmol of amino-polyethylene glycol-carboxyl (molecular weight 2000) in 20 mL of dimethylformamide, add 5 mmol of tert-butyloxycarbonyl hydrazine, 2.4 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 mmol of N-hydroxysuccinimide, react at room temperature for 12 h, add 10 mL of trifluoroacetic acid, stir at room temperature for 2-3 h to obtain hydrazine-terminated polyethylene glycol, react 1 mmol of the obtained hydrazine-terminated polyethylene glycol with 1.2 mmol of N-succinimide ester in 20 mL of 0.1 mol / L acetate-sodium acetate buffer at pH 5 to obtain polyethylene glycol-hydrazone-succinimide ester;

[0060] S2. Weigh 1 mmol of matrix metalloproteinase-sensitive peptide and dissolve it in 20 mL of 0.1 mol / L phosphate buffer at pH 7.4. Add 1.2 mmol of N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester and react at room temperature in the dark to obtain maleimide-terminated peptide.

[0061] S3. Weigh 1 mmol of amphotericin B and 1.5 mmol of 1,6-hexanediamine and dissolve them in 10 mL of dimethylformamide. Add 1.5 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.8 mmol of N-hydroxysuccinimide. React at room temperature in the dark to obtain amphotericin B-amino.

[0062] S4. 1 mmol of amphotericin B-amino obtained in S3 and 1.05 mmol of polyethylene glycol-hydrazone-succinimide ester obtained in S1 were dissolved in 15 mL of dimethylformamide and reacted. The resulting product was then reacted with 1.1 mmol of maleimide-terminated peptide obtained in S2 in 25 mL of phosphate buffer at pH 7.4 in the dark to obtain a dual-response smart linker.

[0063] Preparation Example 2

[0064] The preparation steps of quaternized chitosan are as follows:

[0065] Weigh 10g of chitosan (degree of deacetylation ≥90%, molecular weight 50kDa) and disperse it in 100mL of isopropanol. Slowly add 30mL of a 50% aqueous solution of 2,3-epoxypropyltrimethylammonium chloride while stirring, and react at 70℃ for 12h. The reaction product is precipitated with anhydrous ethanol, washed three times, and dried under vacuum at 45℃ to obtain quaternized chitosan.

[0066] Preparation Example 3

[0067] The preparation steps of catechin-modified hyaluronic acid are as follows:

[0068] Weigh 5.0 g of sodium hyaluronate (molecular weight 200 kDa) and dissolve it in 500 mL of MES buffer at pH 5.0. Add 2.5 g of EDC and 1.5 g of NHS and activate for 30 min. Then add 3.0 g of dopamine hydrochloride and stir at room temperature in the dark for 24 h. Dialyze the reaction solution through a dialysis bag with a molecular weight cutoff of 3500 for 3 days, and freeze-dry to obtain catechol-modified hyaluronic acid.

[0069] Example 1

[0070] A method for preparing soluble hemostatic amphotericin B grafted cotton gauze dressing includes the following steps:

[0071] Step 1: Place a 10×10cm soluble hemostatic cotton gauze sheet in anhydrous ethanol and deionized water for ultrasonic cleaning for 30 minutes each. After drying, immerse 1g of the cleaned cotton gauze in 25mL of 0.5% citric acid aqueous solution. After removing it, squeeze it dry to 100% liquid retention. Crosslink it in a 120℃ oven for 10 minutes, cool it, wash it with deionized water until neutral, dry it, and treat it with low-temperature oxygen plasma (100W) for 5 minutes to obtain the activated cotton gauze, i.e., the base layer.

[0072] Step 2: Immerse 1g of activated cotton yarn obtained in Step 1 in 50mL of MES buffer and activate by shaking at room temperature for 30min. Remove the cotton yarn and immerse it in 100mL of PBS buffer containing 0.05g of dual-response linker at pH 7.4. Add 5mL of polyethylene glycol (molecular weight 1540) and 1mL of Tween-80 as dispersants. Place the system in a constant temperature shaker at 45℃ and react at 100rpm for 6h. Wash 3-4 times with 90% ethanol solution and vacuum dry at 45℃. The dual-response drug graft layer is prepared to obtain AmB grafted cotton yarn.

[0073] Step 3: Prepare quaternized chitosan solution and catechol-modified hyaluronic acid aqueous solution respectively. Using biaxial electrospinning technology, deposit a functional coating on the surface of the AmB grafted cotton yarn obtained in Step 2. Set the feed rate to 0.8 mL / h, voltage to 18 kV, receiving distance to 15 cm, and continue for 30 min. Place the coated cotton yarn in PBS buffer at pH 7.4 and let it stand at 4℃ for 24 h to obtain crude dressing.

[0074] The quaternized chitosan solution is prepared from quaternized chitosan, acetic acid and deionized water, wherein the mass fraction of quaternized chitosan is 2% and the mass fraction of acetic acid is 1%.

[0075] The catechin-modified hyaluronic acid aqueous solution is prepared by catechin-modified hyaluronic acid and deionized water, wherein the mass fraction of catechin-modified hyaluronic acid is 1%.

[0076] Step 4: Rinse the crude dressing obtained in Step 3 3-4 times with PBS buffer at pH 7.4, terminally sterilize with γ-ray (25kGy), seal and package to obtain soluble hemostatic amphotericin B grafted cotton gauze dressing.

[0077] Example 2

[0078] The preparation steps of the dual-responsive linker are as follows:

[0079] S1. Dissolve 2 mmol of amino-polyethylene glycol-carboxyl (molecular weight 2000) in 20 mL of dimethylformamide, add 5 mmol of tert-butyloxycarbonyl hydrazine, 2.4 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 3 mmol of N-hydroxysuccinimide, react at room temperature for 12 h, add 10 mL of trifluoroacetic acid, stir at room temperature for 2-3 h to obtain hydrazine-terminated polyethylene glycol, react 1 mmol of the obtained hydrazine-terminated polyethylene glycol with 1.2 mmol of N-succinimide ester in 20 mL of 0.1 mol / L acetate-sodium acetate buffer at pH 5 to obtain polyethylene glycol-hydrazone-succinimide ester;

[0080] S2. Weigh 1 mmol of matrix metalloproteinase-sensitive peptide and dissolve it in 20 mL of 0.1 mol / L phosphate buffer at pH 7.4. Add 1.2 mmol of N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester and react at room temperature in the dark to obtain maleimide-terminated peptide.

[0081] S3. Weigh 1 mmol of amphotericin B and 1.5 mmol of 1,6-hexanediamine and dissolve them in 10 mL of dimethylformamide. Add 1.5 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.8 mmol of N-hydroxysuccinimide. React at room temperature in the dark to obtain amphotericin B-amino.

[0082] S4. 1 mmol of amphotericin B-amino obtained in S3 and 1 mmol of polyethylene glycol-hydrazone-succinimide ester obtained in S1 were dissolved in 15 mL of dimethylformamide and reacted. The resulting product was then reacted with 1.05 mmol of maleimide-terminated peptide obtained in S2 in 25 mL of phosphate buffer at pH 7.4 in the dark to obtain a dual-response smart linker.

[0083] The difference between this comparative example and Example 1 is that, in step S4, the molar ratio of amphotericin B-amino, polyethylene glycol-hydrazone-succinimide ester, and maleimide-terminated peptide is 1:1:1.05.

[0084] Example 3

[0085] The difference between this comparative example and Example 1 is that, in step three, the mass fraction of catechin-modified hyaluronic acid in the aqueous solution of catechin-modified hyaluronic acid is 2%.

[0086] Comparative Example 1

[0087] The difference between this comparative example and Example 1 is that in step two, a dual-response linker is not used. Instead, 0.05g of amphotericin B is directly dissolved in PBS buffer and physically impregnated with activated cotton yarn.

[0088] Comparative Example 2

[0089] The difference between this comparative example and Example 1 is that in step three, the functional coating is prepared by electrospinning only a 2% by mass solution of ordinary chitosan acetate, without the addition of catechin-modified hyaluronic acid.

[0090] Comparative Example 3

[0091] The difference between this comparative example and Example 1 is that in step one, the citric acid crosslinking treatment of the cotton yarn is omitted, and only plasma activation is performed.

[0092] Comparative Example 4

[0093] The preparation steps of the linker are as follows:

[0094] S1. Weigh 1 mmol of matrix metalloproteinase-sensitive peptide and dissolve it in 20 mL of 0.1 mol / L phosphate buffer at pH 7.4. Add 1.2 mmol of N-succinimide-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester and react at room temperature in the dark to obtain maleimide-terminated peptide.

[0095] S2. Weigh 1 mmol of amphotericin B and 1.5 mmol of 1,6-hexanediamine and dissolve them in 10 mL of dimethylformamide. Add 1.5 mmol of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 1.8 mmol of N-hydroxysuccinimide. React at room temperature in the dark to obtain amphotericin B-amino.

[0096] S4. Dissolve 1 mmol of amphotericin B-amino obtained in S2 and 1.1 mmol of maleimide-terminated peptide obtained in S1 in 25 mL of phosphate buffer solution with pH 7.4 and react in the dark to obtain the linker.

[0097] The difference between this comparative example and Example 1 is that step S1 is omitted, and the pH-responsive unit of polyethylene glycol-hydrazone-succinimide ester is not prepared or introduced in the original S4 step (now S3). Instead, amino-polyethylene glycol-succinimide ester is directly used to react with amphotericin B-amino. In the preparation method of soluble hemostatic amphotericin B grafted cotton gauze dressing, a linker is used.

[0098] test:

[0099] I. Hemostatic properties

[0100] The in vitro whole blood clotting time was measured using the standard YY / T 0693-2008, and the clotting time after the dressing came into contact with fresh human whole blood was recorded.

[0101] II. Drug Release Characteristics

[0102] The dressing samples were placed in pH 7.4 PBS (simulating normal tissue) and pH 6.0 PBS containing MMP-9 (simulating infection environment), and shaken at 37°C. The concentration of amphotericin B released at different time points was determined by HPLC.

[0103] III. Antibacterial Activity

[0104] The samples were processed according to the membrane filtration method in the sterility test method of General Chapter 1101 of the Pharmacopoeia of the People's Republic of China (2020 edition). The minimum inhibitory concentration (MIC) of the dressing extract against Candida albicans (ATCC 10231) was determined by the micro-liquid-based dilution method to evaluate its antifungal efficacy.

[0105] IV. Biocompatibility

[0106] According to GB / T 16886.5-2017 standard, the relative proliferation rate of dressing extract on L929 mouse fibroblasts was determined by MTT assay to evaluate cytotoxicity.

[0107] V. Mechanical Properties

[0108] The tensile strength of the dressing was determined using a universal testing machine (refer to GB / T 24218.3-2010).

[0109] The test results are shown in Table 1.

[0110] Table 1

[0111]

[0112] As shown in Table 1, the dressing prepared in Example 1 exhibits the best overall performance in terms of hemostatic properties, intelligent drug release characteristics, antibacterial activity, biocompatibility, and mechanical properties. The key lies in the design of the dual-response linker, which allows for slow drug release in a normal physiological environment (pH 7.4) (release rate of only 15% over 24 hours), while significantly increasing the release rate to 80% in a simulated infection microenvironment (pH 6.0 + MMP-9), achieving acid / enzyme dual-response drug release. In contrast, Comparative Example 1 (physical impregnation) shows severe burst drug release and lacks long-lasting effect; Comparative Example 4 (without pH-responsive linker) is sluggish in response to acidic environments due to the lack of hydrazone bonds, resulting in a significantly reduced release rate (60%), confirming the necessity of the dual-response mechanism. Furthermore, the excellent hemostatic effect of Example 1 (clotting time 60s) is attributed to the synergistic effect of quaternized chitosan and catechol-modified hyaluronic acid, while the citric acid cross-linked substrate and functional coating jointly ensure its good mechanical properties and biocompatibility. Each comparative example showed shortcomings in its corresponding performance due to the lack of specific processes or components, which proved the rationality and superiority of the complete process design in Example 1.

[0113] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0114] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A process for the preparation of soluble hemostatic amphotericin B grafted cotton yarn dressing characterized by: The method comprises the following steps: Step one, 10*10 cm soluble hemostatic cotton gauze is sequentially cleaned in anhydrous ethanol and deionized water for 30 min, dried, then immersed in a citric acid aqueous solution, rolled dry, crosslinked in an oven at 120 DEG C for 10 min, cooled, washed with deionized water until neutral, dried, and treated with low-temperature oxygen plasma for 5 min to obtain activated cotton gauze, i.e. a base layer; Step two, the activated cotton gauze obtained in step one is immersed in a MES buffer solution, vibrated at room temperature for 30 min, taken out, immersed in a PBS buffer solution containing a double-responsive linker and having a pH of 7.4, and polyethylene glycol and Tween-80 are added as dispersants, the system is placed in a constant-temperature shaking table at 45 DEG C, and reacted at 100 rpm for 6 h, then washed with 90% ethanol solution for 3-4 times, and dried in a vacuum at 45 DEG C to obtain a double-responsive drug grafted layer, thereby obtaining AmB grafted cotton gauze; Step three, quaternary ammonium chitosan solution and catechol-modified hyaluronic acid aqueous solution are respectively prepared, a functional coating is deposited on the surface of the AmB grafted cotton gauze obtained in step two by adopting a double-axis electrospinning technology, the pushing rate is set to 0.8 mL / h, the voltage is 18 kV, the receiving distance is 15 cm, and the deposition is continued for 30 min, then the cotton gauze coated with the coating is placed in a PBS buffer solution having a pH of 7.4, and is statically placed at 4 DEG C for 24 h to obtain a crude product dressing; Step four, the crude product dressing obtained in step three is rinsed with a PBS buffer solution having a pH of 7.4 for 3-4 times, terminal sterilized by adopting gamma rays, sealed and packaged to obtain soluble hemostatic amphotericin B grafted cotton gauze dressing.

2. The process for the preparation of soluble hemostatic amphipathic amphotericin B grafted cotton yarn dressing according to claim 1, characterized by: In step one, the rolling dry operation needs to be rolled to 100% belt liquid rate; the use amount ratio of the cleaned cotton gauze to the citric acid aqueous solution is 1g:(20-30)mL; the mass fraction of the citric acid aqueous solution is 0.5%; and the power of the low-temperature oxygen plasma treatment is 100W.

3. The process for the preparation of soluble hemostatic amphipathic amphotericin B grafted cotton yarn dressing according to claim 2, characterized by: In step two, the use amount ratio of the activated cotton gauze, the MES buffer solution, the double-responsive linker, the buffer solution, the polyethylene glycol, Tween-80 and the 90% ethanol solution is 1g:(40-60)mL:0.05g:100mL:5mL:1mL:(500-1000)mL; the MES buffer solution is a MES buffer solution having a pH of 6 and containing 0.1 mol / L 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 0.05 mol / L N-hydroxysuccinimide (NHS); and the molecular weight of the polyethylene glycol is 1200-1800.

4. The process for the preparation of soluble hemostatic amphipathic amphotericin B grafted cotton yarn dressing as claimed in claim 1, wherein: In step two, the double-responsive linker is prepared according to the following steps: S1, amino-polyethylene glycol-carboxyl is dissolved in dimethylformamide, tert-butyloxycarbonyl hydrazine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added, and the reaction is carried out at room temperature for 12 h, then trifluoroacetic acid is added, and stirring is carried out at room temperature for 2-3 h to obtain hydrazine-terminated polyethylene glycol, and the hydrazine-terminated polyethylene glycol is reacted with N-succinimidyl ester in an acetic acid-sodium acetate buffer solution having a pH of 5 to obtain polyethylene glycol-hydrazone-succinimidyl ester; The amino-polyethylene glycol-carboxyl has a molecular weight of 2000; The amino-polyethylene glycol-carboxyl, tert-butoxycarbonyl hydrazine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and trifluoroacetic acid are in a ratio of 2mmol:20mL:5mmol:2.4mmol:3mmol:10mL; The molar ratio of the hydrazine-terminated polyethylene glycol and N-hydroxysuccinimide ester is 1:1.2, and the usage ratio of the hydrazine-terminated polyethylene glycol and the acetic acid-sodium acetate buffer is 1mmol:20mL; The concentration of the acetic acid-sodium acetate buffer is 0.1mol / L. S2, the matrix metalloproteinase sensitive peptide is weighed and dissolved in a phosphate buffer solution with a pH of 7.4, N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate is added, and the reaction is carried out at room temperature in the dark to obtain a maleimide-terminated peptide; The matrix metalloproteinase sensitive peptide is one of MMP-2 / 9 sensitive peptide, MMP-1 / 13 sensitive peptide and MMP-3 / 7 sensitive peptide; As a preferred technical scheme of the present application, the molar ratio of the matrix metalloproteinase sensitive peptide and N-succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate is 1:1.2; The usage ratio of the matrix metalloproteinase sensitive peptide and the phosphate buffer solution is 1mmol:20mL; The concentration of the phosphate buffer solution is 0.01mol / L. S3, amphotericin B and 1,6-hexanediamine are weighed and dissolved in dimethylformamide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added, and the reaction is carried out at room temperature in the dark to obtain amphotericin B-amino; The molar ratio of the amphotericin B, 1,6-hexanediamine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide is 1:1.5:1.5:1.8; The usage ratio of the amphotericin B and dimethylformamide is 1mmol:10mL. S4, the amphotericin B-amino obtained in S3 is dissolved in the polyethylene glycol-hydrazone-succinimide ester obtained in S1 to react, and then the obtained product is dissolved in the maleimide-terminated peptide obtained in S2 in a phosphate buffer solution with a pH of 7.4 to react in the dark to obtain a double-response intelligent linker; The molar ratio of the amphotericin B-amino and the polyethylene glycol-hydrazone-succinimide ester is 1:1.05; The usage ratio of the amphotericin B-amino and dimethylformamide is 1mmol:15mL; The molar ratio of the polyethylene glycol-hydrazone-succinimide ester and the maleimide-terminated peptide is 1:1.05; The usage ratio of the polyethylene glycol-hydrazone-succinimide ester and the phosphate buffer solution is 1mmol:25mL.

5. The process for the preparation of soluble hemostatic amphipathic amphotericin B grafted cotton yarn dressing as claimed in claim 1 wherein: In step three, the quaternary ammonium chitosan solution is configured from quaternary ammonium chitosan, acetic acid and deionized water, wherein the mass fraction of quaternary ammonium chitosan is 2%, and the mass fraction of acetic acid is 1%.

6. The process for the preparation of soluble hemostatic amphotericin B grafted cotton yarn dressing as claimed in claim 1, wherein: In the third step, the catechol-modified hyaluronic acid aqueous solution is prepared by mixing the catechol-modified hyaluronic acid and deionized water, wherein the mass fraction of the catechol-modified hyaluronic acid is 1%.

7. The process for the preparation of soluble hemostatic amphotericin B grafted cotton yarn dressing as claimed in claim 1 wherein: In the third step, the preparation of the quaternary ammonium chitosan is as follows: 10g of chitosan is dispersed in 100mL of isopropyl alcohol, and then 30mL of 2,3-epoxypropyl trimethyl ammonium chloride aqueous solution with a mass fraction of 50% is slowly added under stirring, and the mixture is reacted at 70℃ for 12h, precipitated with anhydrous ethanol, washed for 3-4 times, and dried at 45℃ under vacuum to obtain the quaternary ammonium chitosan. As a preferred technical solution of the present application, the degree of deacetylation of the chitosan is greater than or equal to 90%, and the molecular weight is 50kDa.

8. The process for the preparation of soluble hemostatic amphipathic amphotericin B grafted cotton yarn dressing as claimed in claim 1 wherein: In the third step, the preparation of the catechol-modified hyaluronic acid is as follows: Sodium hyaluronate is weighed and dissolved in MES buffer with a pH of 5.0, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide are added, and activated for 30min, then dopamine hydrochloride is added, and the mixture is stirred at room temperature in the dark for 24h, the obtained reaction solution is dialyzed in a dialysis bag with a molecular weight cut-off of 3500 for 3 days, and then freeze-dried to obtain the catechol-modified hyaluronic acid. As a preferred technical solution of the present application, the molecular weight of the sodium hyaluronate is 200kDa. As a preferred technical solution of the present application, the ratio of the amounts of the sodium hyaluronate, MES buffer, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide and dopamine hydrochloride is 5g:500mL:2.5g:1.5g:3.0g.

9. The process for the preparation of soluble hemostatic amphotericin B grafted cotton yarn dressing as claimed in claim 1 wherein: In the fourth step, the dose of the gamma rays is 25kGy.

10. The soluble hemostatic amphotericin B grafted cotton yarn dressing prepared by the method according to any one of claims 1-9.

10. The soluble hemostatic amphotericin B grafted cotton yarn dressing prepared by the method according to any one of claims 1-9.

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