An injectable antibacterial hemostatic hydrogel dressing, its preparation method and application

Abstract

The present invention discloses an injectable antibacterial hemostatic hydrogel dressing and its preparation method and application. The raw materials for its preparation have excellent biocompatibility, and the hydrogen peroxide / horseradish peroxidase catalytic oxidation system adopted can provide a mild, efficient and highly biocompatible cross-linking process for the hydrogel dressing. The hydrogel dressing prepared by this method has excellent antibacterial and antioxidant properties, high strength and high tissue adhesion strength, and has an excellent hemostatic effect, and can be well applied to the field of skin trauma treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and particularly relates to an injectable antibacterial hemostatic hydrogel dressing, a preparation method thereof and an application thereof. Background Art

[0002] The skin is the largest organ of the human body and is also an organ with very active metabolism, which is crucial for maintaining the body's internal balance and protecting the body. At the same time, the skin is also the most vulnerable tissue. According to statistics, millions of people need to have their wounds caused by surgery or trauma closed every year. At present, the main means of wound closure clinically used are surgical suture and surgical stapling, but these traditional treatment methods have many disadvantages, such as causing secondary damage to human tissues, triggering inflammation, some requiring subsequent removal, and generating scar tissue that affects appearance. Biomedical glue is a type of wound repair material with functions such as sealing the wound surface and promoting wound healing. Among many medical glues, injectable antibacterial hemostatic hydrogel dressings have become one of the more ideal alternative biomedical glues because they can quickly seal the wound surface, maintain a moist wound environment, cool the wound surface, allow oxygen to permeate and promote wound healing, etc.

[0003] Due to the actual advantages of biomedical hydrogel dressings, a large number of products have been developed. However, there are still some deficiencies in the performance of existing hydrogel dressings: 1) The adhesion strength of injectable antibacterial hemostatic hydrogel dressings is relatively low; 2) The biocompatibility of high-strength hydrogel dressings is not good; 3) The bioactivity for promoting skin tissue repair and regeneration is insufficient.

[0004] Polyethylene glycol is a type of synthetic polymer formed by the stepwise addition polymerization of ethylene oxide with water or ethylene glycol. The terminal hydroxyl groups make it easy to be further modified to optimize its physical and chemical properties, mechanical properties, etc. The biomedical hydrogel dressings developed based on polyethylene glycol have good biocompatibility. They usually do not cause immune rejection, have a modulus similar to that of human soft tissues, and exhibit good adhesion to the surface of wet tissues. The disadvantage of such adhesives is that the swelling rate is relatively high, and the cohesive force in the hydrogel dressing rapidly decreases in the water absorption equilibrium state, greatly limiting its tissue adhesion performance.

[0005] Mussels are a common type of bivalve marine mollusk that can produce a strong adhesion to underwater structures. Inspired by this, hydrogel dressings based on catechol groups have been developed. For catechol-functionalized polymer chains, in terms of chemical crosslinking, in the past, when preparing hydrogel dressings from most catechol-modified polymers, sodium periodate was used as an oxidative coupling agent. This strong oxidant and its reduction products both have relatively high toxicity and poor biocompatibility; in terms of physical crosslinking, the gels with catechol groups crosslinked by iron ions are sensitive to acids, the hydrogel dressings are extremely unstable, and excessive iron ions will produce certain cytotoxicity. Summary of the Invention

[0006] The object of the present invention is to overcome the disadvantages of the above-mentioned prior art, and to provide an injectable antibacterial hemostatic hydrogel dressing, its preparation method and application, so as to solve the problems of low adhesion strength and poor biocompatibility of hydrogels in the prior art.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] A preparation method of an injectable antibacterial hemostatic hydrogel dressing is to mix an aqueous solution of CPD and an aqueous solution of an amino-modified end-hydroxyl triblock amphiphilic polymer, adjust the pH value of the mixed system to 6-8, and then add hydrogen peroxide and an aqueous solution of horseradish peroxidase. After oxidative crosslinking, a polymer network is formed to obtain the hydrogel dressing.

[0009] The preparation process of CPD in the aqueous solution of CPD is as follows: citric acid, polyethylene glycol and dopamine hydrochloride are mixed in a reaction vessel to form a first reaction system; under the environment of introducing nitrogen, the first reaction system is stirred at a first set temperature to make the first reaction system form a molten state of the first reaction system; the temperature of the molten state of the first reaction system is lowered to a second set temperature, and the molten state of the first reaction system is stirred at the second set temperature to react to obtain a reaction product. After dialysis of the reaction product, the dialysis product is rotary evaporated and freeze-dried to obtain CPD.

[0010] The preparation process of the amino-modified end-hydroxyl triblock amphiphilic polymer in the aqueous solution of the amino-modified end-hydroxyl triblock amphiphilic polymer is as follows: the end-hydroxyl triblock amphiphilic polymer is heated to melt, the molten end-hydroxyl triblock amphiphilic polymer is dried, and the dried end-hydroxyl triblock amphiphilic polymer is dissolved in dichloromethane to form a second reaction system; N,N'-carbonyldiimidazole is added to dichloromethane to form a third reaction system; the third reaction system is dropped into the second reaction system to form a fourth reaction system, ethylenediamine is added to the fourth reaction system, stirred and then water is added to form a fifth reaction system. After the fifth reaction system is extracted with dichloromethane, the extraction product is washed, the precipitate obtained by washing is dissolved in an organic solvent, centrifuged and separated, and the centrifuged product is dried to obtain the amino-modified end-hydroxyl triblock amphiphilic polymer.

[0011] A further improvement of the present invention lies in:

[0012] Preferably, the concentration of the CPD aqueous solution is 500 mg / mL, the concentration of the amino-modified terminal hydroxyl triblock amphiphilic polymer aqueous solution is 300 mg / mL, and the mixing molar ratio of the CPD aqueous solution and the amino-modified terminal hydroxyl triblock amphiphilic polymer aqueous solution is 4:5.

[0013] Preferably, 5 - 25 μL of hydrogen peroxide is added to 1 mL of the mixed system, and 40 μL of horseradish peroxidase aqueous solution is added to 1 mL of the mixed system;

[0014] The mixing molar ratio of the citric acid, polyethylene glycol, and dopamine hydrochloride is 1.1:1:0.5, the first set temperature is 160 °C, and the second set temperature is 140 °C.

[0015] Preferably, the CPD aqueous solution and the amino-modified terminal hydroxyl triblock amphiphilic polymer aqueous solution are pre-cooled and then mixed to form a mixed system.

[0016] Preferably, the first reaction system in a molten state is stirred at the second set temperature for 48 h.

[0017] Preferably, the terminal hydroxyl triblock amphiphilic polymer is Pluronic F68, Pluronic F127, Pluronic F188, polyethylene glycol-b-polylactic acid-b-polyethylene glycol, or polyethylene glycol-b-polycaprolactone-b-polyethylene glycol.

[0018] Preferably, the preparation process of the amino-modified terminal hydroxyl triblock amphiphilic polymer is as follows:

[0019] (2A) The terminal hydroxyl triblock amphiphilic polymer is heated and stirred to melt at 80 °C under a nitrogen stream, the nitrogen stream is cut off, and vacuum is started for 12 hours to dry the molten terminal hydroxyl triblock amphiphilic polymer;

[0020] (2B) Under a nitrogen stream, the dried terminal hydroxyl triblock amphiphilic polymer is dissolved in dichloromethane under an ice-water bath condition to form a second reaction system. The temperature of the ice-water bath is less than 4 °C, and the concentration of the second reaction system is 50 mM;

[0021] (2C) N,N'-carbonyldiimidazole is dissolved in dichloromethane to form a third reaction system. The concentration of the third reaction system is 800 mM. The third reaction system is added dropwise to the second reaction system under a nitrogen stream to form a fourth reaction system. The mixing volume ratio of the third reaction system and the second reaction system is 5:4;

[0022] (2D) Add ethylenediamine dropwise to the fourth reaction system under a nitrogen stream and stir to mix evenly to form a fifth reaction system, where the molar ratio of ethylenediamine to the hydroxyl-terminated triblock amphiphilic polymer is 100:1;

[0023] (2E) Add deionized water to the fifth reaction system, extract with dichloromethane, wash with saturated brine, pre-cool the precipitate obtained from the washing in ether at -80 °C, and centrifuge to separate the precipitant and the product at a rotation speed of 8000 rpm. After drying, the final product can be obtained.

[0024] An injectable antibacterial and hemostatic hydrogel dressing prepared by the preparation method described in any one of the above, comprising an amino-modified hydroxyl-terminated triblock amphiphilic copolymer and CPD; the amino-modified hydroxyl-terminated triblock amphiphilic copolymers are crosslinked with each other, and the amino-modified hydroxyl-terminated triblock amphiphilic copolymer and CPD are crosslinked through Schiff base or Michael addition.

[0025] Preferably, the amino-modified hydroxyl-terminated triblock amphiphilic copolymer is loaded with a hydrophobic drug.

[0026] An application of the injectable antibacterial and hemostatic hydrogel dressing described above, characterized in that the hydrogel dressing is used for full-thickness wound repair, hemostasis, antibacterial or drug carrier.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention discloses a preparation method of an injectable antibacterial and hemostatic hydrogel dressing. The raw materials used in this preparation method have excellent biocompatibility. The hydrogen peroxide / horseradish peroxidase catalytic oxidation system adopted can provide a mild, efficient and highly biocompatible crosslinking process for the hydrogel dressing. The hydrogel dressing prepared by this method has excellent antibacterial and antioxidant properties, high strength and high tissue adhesion strength, and excellent hemostatic effect, and can be well applied to the field of skin trauma treatment.

[0029] (1) The component CPD is rich in catechol groups. First, under the action of an oxidant, the catechol groups can be oxidized to quinone groups, which play a role in crosslinking the polymer network through forms such as Schiff base and Michael addition; furthermore, the catechol groups can covalently couple with amino groups, sulfhydryl groups, etc. on the tissue surface under the action of an oxidant, providing considerable tissue adhesion strength; finally, the catechol groups have good reducibility, can effectively absorb oxygen free radicals at the wound site, and can effectively avoid problems such as lipid peroxidation, DNA double helix chain breakage and enzyme inactivation caused by oxidative stress, further promoting scarless repair.

[0030] (2) The amino - terminated hydroxyl - terminated triblock amphiphilic polymer (such as PF127 - NH2) introduced into the hydrogel dressing network, firstly, as a typical hydroxyl - terminated triblock amphiphilic copolymer, it can self - assemble into micelles in an aqueous environment, and with the assistance of hydrophobic interactions, it provides a mechanical dissipation mechanism for the hydrogel dressing network, significantly improving the cohesion and mechanical properties of the hydrogel dressing; secondly, the micelles formed by self - assembly can be used to load hydrophobic drugs, such as astragaloside IV, which can further promote scar - free repair; finally, the amino modification can undergo a Schiff base reaction with the catechol groups on the CPD segment to produce a stable interpenetrating network covalent cross - linking effect, forming an interpenetrating network to further enhance the mechanical properties of the hydrogel dressing.

[0031] (3) At the same time, using CPD and the hydroxyl - terminated triblock amphiphilic copolymer as the two components of the hydrogel, in addition to showing the respective performance advantages of their own bulk, there are also obvious mutual performance enhancement effects: First, the poly(ethylene glycol) - based CPD single - component hydrogel is usually extremely prone to swelling. When the hydroxyl - terminated triblock amphiphilic copolymer is introduced, this copolymer self - assembles into micelles in an aqueous solution and cross - links with CPD. The formed double - component hydrogel can effectively improve the defect of the CPD single - component hydrogel being prone to swelling, and can reduce the maximum swelling ratio to less than 250%; Second, the biggest defect of the single - component hydrogel formed by the hydroxyl - terminated triblock amphiphilic copolymer (such as PF127) is the serious lack of tissue adhesion. During application, it may fall off prematurely and fail to achieve a sealing effect. When introducing CPD rich in catechol, it can provide extremely excellent tissue adhesion to overcome the inherent defect of the single - component hydrogel of the hydroxyl - terminated triblock amphiphilic copolymer.

[0032] (4) The hydrogen peroxide / horseradish peroxidase catalytic oxidation system used not only overcomes the biosafety problems and mechanical property instability problems brought by traditional oxidation methods, but also provides a mild, efficient and highly biocompatible cross - linking process for the hydrogel dressing.

[0033] (5) The use of hydrogen peroxide not only overcomes the disadvantages of traditional cross - linkers, but also provides excellent antibacterial effects for the hydrogel dressing, and can produce obvious growth inhibitory effects on Pseudomonas aeruginosa (PA) and Methicillin - resistant Staphylococcus aureus (MRSA).

[0034] (6) In daily disinfection products, medical hydrogen peroxide has been widely used. Hydrogen peroxide, as its main component, is a good oxygen - free radical donor. Under reasonable catalytic conditions, the hydrogen peroxide / horseradish peroxidase catalytic oxidation system can provide a rapid oxidation cross - linking mechanism for polymers functionalized with catechol groups, and can gel the hydrogel dressing rapidly in 50 s, and can be effectively applied to hemostasis in parts such as the liver and veins.

[0035] The present invention also discloses an injectable antibacterial hemostatic hydrogel dressing and its application. The rheological properties, mechanical strength, swelling ratio, tissue adhesion strength, DPPH free radical scavenging rate, blood compatibility, cytotoxicity, etc. of the hydrogel dressing prepared by the present invention can be adjusted by regulating the dosage of hydrogen peroxide, and the component ratio of the hydrogel dressing can be screened and optimized accordingly. Through experiments on a rat dorsal full-thickness skin injury repair model, antibacterial zone and release antibacterial experiments, a mouse liver model and a rabbit femoral vein hemostasis model, it is verified that the hydrogel dressing prepared by the present invention can exhibit excellent performance in aspects such as full-thickness skin wound repair, antibacterial, and hemostasis. Brief Description of the Drawings

[0036] Figure 1 is a schematic diagram of the mechanism for enhancing the mechanical properties of the hydrogel dressing;

[0037] Figure 2(a) is a shear modulus curve graph of the hydrogel dressing; Figure 2(b) is an axial compression stress-strain curve graph of the hydrogel dressing; Figure 2(c) is an adhesion strength graph of the hydrogel dressing to porcine skin; Figure 2(d) is an adhesion strength graph of the hydrogel dressing to porcine skeletal muscle.

[0038] Figure 3(a) is a swelling ratio curve graph of the hydrogel dressing; Figure 3(b) is a DPPH free radical scavenging rate graph of the hydrogel dressing;

[0039] Figure 4(a) is a hemolytic activity graph of the hydrogel dressing after preparation; Figure 4(b) is a hemolytic activity graph of the hydrogel dressing after 48-hour dialysis; Figure 4(c) is a cell viability graph measured from the leachate of the hydrogel dressing; Figure 4(d) is a cell viability graph measured by the contact method of the hydrogel dressing after preparation; Figure 4(e) is a cell viability graph measured by the contact method of the hydrogel dressing after 48-hour dialysis; Figure 4(f) is a statistical graph of the antibacterial zone diameter of the hydrogel dressing against Pseudomonas aeruginosa; Figure 4(g) is a statistical graph of the antibacterial zone diameter of the hydrogel dressing against methicillin-resistant Staphylococcus aureus; Figure 4(h) is a graph of the antibacterial release characterization results of the hydrogel dressing.

[0040] Figure 5(a) is a graph of the blood loss in the mouse liver hemostasis model of the hydrogel dressing; Figure 5(b) is a graph of the hemostasis time in the mouse liver hemostasis model of the hydrogel dressing; Figure 5(c) is a graph of the blood loss in the rabbit femoral vein hemostasis model of the hydrogel dressing; Figure 5(d) is a graph of the hemostasis time in the rabbit femoral vein hemostasis model of the hydrogel dressing.

[0041] Figure 6(a) is a macroscopic view of the rat dorsal full-thickness skin wound repair by the hydrogel dressing (the scale represents 5 mm); Figure 6(b) is a hematoxylin-eosin stained tissue section of the rat dorsal full-thickness skin wound repair by the hydrogel dressing (the scale represents 500 μm); Figure 6(c) is a Masson stained tissue section of the rat dorsal full-thickness skin wound repair by the hydrogel dressing (the scale represents 500 μm).

[0042] Figure 7 Figure 2 is the network structure diagram of the prepared hydrogel. DETAILED DESCRIPTION

[0043] The present invention is further described in detail below with reference to the accompanying drawings and specific embodiments:

[0044] The preparation method of the injectable antibacterial hemostatic hydrogel dressing having high strength and tissue adhesion strength of the present invention comprises the following steps:

[0045] (1) synthesizing the high molecular weight copolymer poly(ethylene glycol, citrate) ester-g-dopamine (CPD) in one step by high temperature melt polymerization;

[0046] The synthesis of the high molecular weight copolymer poly (polyethylene glycol, citrate) ester-g-dopamine comprises the following steps:

[0047] (1A) placing citric acid, polyethylene glycol and dopamine hydrochloride in a molar ratio of 1.1:1:0.5 into a reaction vessel at once;

[0048] (1B) After nitrogen gas was introduced into the reaction vessel for 10 minutes to expel the air in the flask, the mixture was heated at 160° C. and stirred for 3 hours under a nitrogen flow to completely melt the citric acid, polyethylene glycol and dopamine hydrochloride mixture. The nitrogen flow prevented air from entering the entire reaction vessel, thereby oxidizing dopamine.

[0049] (1C) Stop introducing nitrogen into the reaction vessel and gradually reduce the reaction temperature to 140° C. to prevent the high temperature from destroying dopamine for too long and causing dopamine to be oxidized.

[0050] (1D) After stirring for 48 hours at 140 °C, the reaction was stopped to generate CPD. The CPD was dissolved in deionized water and prepared into a solution with a concentration of 200 mg / mL. The solution was then dialyzed in deionized water for 72 hours using a dialysis bag with a molecular weight cutoff of 1000. Finally, the final product CPD was obtained by rotary evaporation and freeze drying.

[0051] (2) Amino modification of both ends of the hydroxyl-terminated triblock amphiphilic polymer, such as using N,N'-carbonyldiimidazole (CDI) to activate the terminal hydroxyl groups of Pluronic F127 (PF127), and modifying the two ends of the activated PF127 with ethylenediamine to synthesize amino-terminated Pluronic F127 (PF127-NH2);

[0052] The two ends of the hydroxyl-terminated triblock amphiphilic polymer are hydrophilic chain segments, and the middle segment is a hydrophobic chain segment, such as Pluronic F68, Pluronic F127, Pluronic F188, polyethylene glycol-b-poly(lactic acid)-b-polyethylene glycol, polyethylene glycol-b-poly(ε-caprolactone)-b-polyethylene glycol;

[0053] The preparation steps of the amino-terminated hydroxyl-terminated triblock amphiphilic polymer include:

[0054] (2A) Heat the hydroxyl-terminated triblock amphiphilic polymer under a nitrogen stream at 80 °C with continuous stirring for 3 hours until it is completely melted. Then cut off the nitrogen stream and start vacuum pumping for 12 hours to dry and remove water from the melted hydroxyl-terminated triblock amphiphilic polymer;

[0055] (2B) Dissolve the dried hydroxyl-terminated triblock amphiphilic polymer in dichloromethane in an ice-water bath (the interaction between hydrophilic chain segments weakens at low temperature, which can accelerate dissolution). The temperature of the ice-water bath is less than 4 °C, and its concentration is 50 mM;

[0056] (2C) Dissolve N,N'-carbonyldiimidazole (CDI) in dichloromethane to a concentration of 800 mM. After the hydroxyl-terminated triblock amphiphilic polymer is completely dissolved, add it dropwise to the system described in (2B) at a volume ratio of 5:4 under a nitrogen stream and continuously stir and react for 12 hours;

[0057] (2D) Add ethylenediamine dropwise to the system described in (2C) at a molar ratio of ethylenediamine to hydroxyl-terminated triblock amphiphilic polymer of 100:1 under a nitrogen stream and continuously stir and react at room temperature for 24 hours;

[0058] (2E) Add 300 mL of deionized water to the system described in (2D) at one time, then extract with dichloromethane, wash with saturated brine, and finally precipitate it into diethyl ether pre-cooled to -80 °C, and centrifuge at a speed of 8000 rpm to separate the precipitant and the product. After drying, the final product can be obtained.

[0059] (3) Use the oxidation catalytic system of hydrogen peroxide / horseradish peroxidase to crosslink CPD and the amino-modified hydroxyl-terminated triblock amphiphilic polymer (such as PF127-NH2) to form a polymer network, thereby obtaining an injectable antibacterial hemostatic hydrogel dressing with high strength and tissue adhesion strength.

[0060] The preparation steps of the injectable antibacterial hemostatic hydrogel dressing with high strength and tissue adhesion strength include:

[0061] (3A) Prepare an aqueous solution of CPD using PBS buffer as the solvent at a concentration of 500 mg / mL, prepare an aqueous solution of an amino-modified triblock amphiphilic polymer (such as PF127-NH2) using PBS buffer as the solvent at a concentration of 300 mg / mL, prepare an aqueous solution of horseradish peroxidase using PBS buffer as the solvent at a concentration of 5 mg / mL, and place all of them in an ice-water mixture for pre-cooling; the triblock amphiphilic polymer with terminal hydroxyl groups is pre-cooled to a flowing state.

[0062] (3B) Mix the pre-cooled aqueous solution of CPD and the aqueous solution of the amino-modified triblock amphiphilic polymer (such as PF127-NH2) in a ratio of 4:5 and make the components uniform; each 1 mL of the hydrogel dressing system contains 400 μL of the aqueous solution of CPD; each 1 mL of the hydrogel dressing system contains 500 μL of the aqueous solution of the triblock amphiphilic polymer with terminal hydroxyl groups.

[0063] (3C) Use 10 M aqueous sodium hydroxide solution to adjust the pH value of the mixed system described in (3B) to 6 - 8.

[0064] (3D) Add hydrogen peroxide and the aqueous solution of horseradish peroxidase to the system with neutral pH adjusted in (3C) in sequence for oxidative crosslinking to form a polymer network. (After mixing), each 1 mL of the hydrogel dressing system requires 5 - 25 μL of hydrogen peroxide and 40 μL of the aqueous solution of horseradish peroxidase.

[0065] The present invention prepares an injectable antibacterial hemostatic hydrogel dressing with high strength and tissue adhesion strength, which can be used as a wound dressing in aspects such as full-thickness wound repair, hemostasis, antibacterial, and drug carrier.

[0066] An injectable antibacterial hemostatic hydrogel dressing with high strength and tissue adhesion strength is prepared by the preparation method as described above;

[0067] An injectable antibacterial hemostatic hydrogel dressing with high strength and tissue adhesion strength prepared by the preparation method as described above is used as a wound dressing in the applications such as full-thickness wound repair, hemostasis, antibacterial, and drug carrier.

[0068] The present invention is further described in detail below with specific examples, which are explanations of the present invention rather than limitations.

[0069] Example 1

[0070] (1) 6.687 g of citric acid (purchased from Vetec), 12.658 g of polyethylene glycol (number average molecular weight = 400, purchased from Aladdin), and 3 g of dopamine hydrochloride (purchased from Energy Chemical) were placed in a 50 mL round-bottom flask and purged with nitrogen for ten minutes. Subsequently, it was placed in a preheated oil bath at 160 °C and continuously stirred and heated for melting for 3 hours. When the entire reaction system became clear, i.e., in a completely molten state, the temperature of the oil bath was lowered to 140 °C. After the temperature drop was completed, the nitrogen purge was stopped, and a positive vacuum was applied to cause condensation polymerization for 48 hours. The prepared poly(polyethylene glycol, citric acid) ester-g-dopamine (CPD) was dissolved in deionized water to prepare a solution with a concentration of 200 mg / mL. Subsequently, it was dialyzed in deionized water using a dialysis bag with a molecular weight cut-off of 1000 for 72 hours. Finally, rotary evaporation and freeze-drying were performed to obtain the final product.

[0071] (2) 1 mmol of PF127 (12.7 g) was placed in a 50 mL three-necked flask and purged with nitrogen for 10 min. Then it was placed in a preheated oil bath at 80 °C and heated for melting for 3 hours. When it was completely molten and clear, the nitrogen flow was cut off, and a vacuum was applied for drying and water removal for 12 hours. 20 mL of dichloromethane was added to the dried PF127, and it was stirred in an ice-water bath for 30 minutes under a nitrogen flow until completely dissolved. Subsequently, 3.24 g of N,N'-carbonyldiimidazole (CDI) was dissolved in 20 mL of dichloromethane and added dropwise into the three-necked flask under a nitrogen flow. The reaction was carried out for 12 hours, and then 10 mL of ethylenediamine was added dropwise into the system and the reaction continued for 24 hours. 300 mL of deionized water was added to the reaction-terminated system and shaken well. Subsequently, it was extracted with dichloromethane three times, with 250 mL of dichloromethane used each time. Then the organic extract was washed with saturated brine, with 250 mL of saturated brine used each time. After the washing and purification were completed, a large amount of dichloromethane was evaporated using a rotary evaporator, and the remaining solution was precipitated using -80 °C pre-cooled ether as a precipitant. The precipitate system was centrifuged at 8000 rpm to separate the precipitate, which was then placed in a vacuum drying oven for vacuum drying to obtain the final product.

[0072] (3) Dissolve the CPD polymer in PBS buffer at a concentration of 500 mg / mL, dissolve PF127-NH2 in PBS buffer at a concentration of 300 mg / mL, dissolve horseradish peroxidase in deionized water at a concentration of 5 mg / mL, and prepare a 10 M NaOH solution. First, fully pre-cool the above solutions and 30% hydrogen peroxide in an ice-water bath. Take 400 μL of the pre-cooled CPD solution and 400 μL of the pre-cooled PF127-NH2 solution and mix them evenly in a 4 mL centrifuge tube. Then add 5 μL of the 10 M NaOH solution and mix evenly to make the reaction system neutral. Add 5 μL of 30% hydrogen peroxide to the neutralized reaction system and add a certain amount of PBS buffer so that the total volume of hydrogen peroxide and PBS buffer is 55 mL. Mix evenly again and place it in an ice-water bath for full pre-cooling. Finally, add 40 μL of the horseradish peroxidase aqueous solution to the system so that the entire reaction system is oxidized and cross-linked into a hydrogel dressing, which is named CPD / PF127-NH2@H2O2 5.

[0073] Example 2

[0074] Different from Example 1, the dosage of hydrogen peroxide in step (3) was adjusted from 5 μL to 10 μL, and the prepared hydrogel dressing was named CPD / PF127-NH2@H2O2 10.

[0075] Example 3

[0076] Different from Example 1, the dosage of hydrogen peroxide in step (3) was adjusted from 5 μL to 15 μL, and the prepared hydrogel dressing was named CPD / PF127-NH2@H2O2 15.

[0077] Example 4

[0078] Different from Example 1, the dosage of hydrogen peroxide in step (3) was adjusted from 5 μL to 20 μL, and the prepared hydrogel dressing was named CPD / PF127-NH2@H2O2 20.

[0079] Example 5

[0080] Different from Example 1, the dosage of hydrogen peroxide in step (3) was adjusted from 5 μL to 25 μL, and the prepared hydrogel dressing was named CPD / PF127-NH2@H2O2 25.

[0081] Example 6

[0082] Different from Example 1, 500 μL of PF127-NH2 solution in step (3) was replaced with 500 μL of PBS buffer, and at the same time, the dosage of hydrogen peroxide was adjusted from 5 μL to 10 μL. The prepared hydrogel dressing was named CPD / PBS@H2O220.

[0083] The hydrogel dressings prepared by the present invention have excellent properties in terms of rheological properties, mechanical strength, swelling ratio, tissue adhesion strength, DPPH free radical scavenging rate, blood compatibility, cytotoxicity, etc. Through experiments on a full-thickness skin injury repair model on the back of rats, antibacterial zone and release antibacterial experiments, mouse liver model and rabbit femoral vein hemostasis model experiments, it is verified that the hydrogel dressings prepared by the present invention can show sufficient superiority in full-thickness skin wound repair, antibacterial, hemostasis, etc. The following is a detailed analysis in combination with the attached drawings and experimental data.

[0084] Figure 1 It is a schematic diagram of the mechanical property enhancement mechanism of the hydrogel dressing prepared by the present invention. When subjected to external compressive stress, the micelles self-assembled by PF127 can provide a good mechanical dissipation mechanism to improve the mechanical properties of the hydrogel dressing;

[0085] Figure 2(a) is a shear modulus curve graph of the hydrogel dressing prepared by the present invention. Generally, as the hydrogen peroxide content increases, its storage modulus becomes higher and higher. For the preferred sample CPD / PF127-NH2@H2O220, its storage modulus (G’) can reach 100 kPa, which can provide sufficient cohesion for the hydrogel dressing; from the intersection of the storage modulus (G’) and the loss modulus (G”), and the time required for it to crosslink into a hydrogel dressing is getting shorter and shorter. For CPD / PF127-NH2@H2O210, CPD / PF127-NH2@H2O215, CPD / PF127-NH2@H2O220, CPD / PF127-NH2@H2O225, the required time is about 50 seconds, which can meet the requirement of quickly closing the wound during the hemostasis process;

[0086] Figure 2(b) is an axial compression stress-strain curve graph of the hydrogel dressing prepared by the present invention. It can be seen from the figure that when the axial compression strain reaches 60%, its axial stress is higher than 100 kPa;

[0087] Figure 2(c) is an adhesion strength graph of the hydrogel dressing prepared by the present invention to pig skin. It can be seen from the figure that the hydrogel dressing prepared by the present invention shows excellent adhesion strength. In particular, for the preferred sample CPD / PF127-NH2@H2O220, its shear adhesion strength to pig skin can reach about 22 kPa, which can be attributed to the covalent cross-linking of the residual catechol groups in the hydrogel dressing network with a large number of free amino groups, carboxyl groups, tyrosine residue sulfhydryl groups, etc. on the skin surface during in-situ gelation;

[0088] Figure 2(d) is the adhesion strength diagram of the hydrogel dressing prepared by the present invention to porcine skeletal muscle. It can be seen from the figure that in a high-humidity environment, its tissue adhesion strength is still superior to that of commercial fibrin glue adhesives ( 5 kPa).

[0089] Figure 3(a) is the swelling rate curve diagram of the hydrogel dressing prepared by the present invention. It can be seen from the figure that as the hydrogen peroxide content increases, its swelling rate increases. However, for the preferred sample CPD / PF127-NH2@H2O2 20, its swelling rate is only about 100%, which is greatly improved compared with the swelling behavior of other polyethylene glycol-based hydrogel dressings, ensuring that the hydrogel dressing maintains strong gel cohesion in a high-humidity environment, such as during the hemostasis process;

[0090] Figure 3(b) is the DPPH free radical scavenging rate diagram of the hydrogel dressing prepared by the present invention. It can be seen from the figure that the hydrogel dressings prepared by the present invention all have good free radical scavenging ability and can well promote scarless repair;

[0091] Figure 4(a) 、 4(b) are the hemolytic activity diagrams of the hydrogel dressing prepared by the present invention after preparation and after 48-hour dialysis, respectively. It can be seen from the figure that the hydrogel dressing prepared by the present invention does not have hemolytic activity after two days;

[0092] Figure 4(c) is the cell viability diagram measured from the leachate of the hydrogel dressing prepared by the present invention. It can be seen from the figure that in this test model, the hydrogel dressing prepared by the present invention has no cytotoxicity to mouse fibroblasts (L929);

[0093] Figure 4(d) 、 4(e) are the cell viability diagrams measured by the contact method of the hydrogel dressing prepared by the present invention after preparation and after 48-hour dialysis, respectively. It can be seen that after two days of exerting the bactericidal function, it has no cytotoxicity to mouse fibroblasts (L929);

[0094] Figure 4(f) 、 4(g) are the statistical diagrams of the inhibition zone diameters of the hydrogel dressing prepared by the present invention against Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus, respectively. It can be seen from the figure that for CPD / PF127-NH2@H2O2 20 and CPD / PF127-NH2@H2O2 25, the hydrogen peroxide contained in them can achieve an effective diffusion bactericidal effect in a solid medium;

[0095] Figure 4(h) is the antibacterial characterization result diagram of the hydrogel dressing. It can be seen from the figure that for CPD / PF127-NH2@H2O220 and CPD / PF127-NH2@H2O225, the hydrogen peroxide contained in them can achieve an effective bactericidal effect through release.

[0096] Figure 5(a) 、 5(b) They are respectively the blood loss diagram and the hemostasis time diagram of the mouse liver hemostasis model of the hydrogel dressing prepared by the present invention. It can be seen from the figure that the hydrogel dressing prepared by the present invention can effectively reduce the blood loss during liver bleeding and achieve the hemostasis effect in a short time;

[0097] Figure 5(c) and Figure 5(d) are respectively the blood loss diagram and the hemostasis time diagram of the rabbit femoral vein hemostasis model of the hydrogel dressing prepared by the present invention. It can be seen from the figure that the hydrogel dressing prepared by the present invention can effectively reduce the blood loss during rabbit venous bleeding and achieve the hemostasis effect in a short time;

[0098] Figure 6(a), Figure 6(b), and Figure 6(c) are respectively the macroscopic diagram of the full-thickness dorsal skin wound repair of rats, the hematoxylin-eosin staining diagram of tissue sections, and the Masson staining diagram of tissue sections of the hydrogel dressing prepared by the present invention. It can be seen from the figure that compared with the other control groups, the CPD / PF127-NH2@H2O220 prepared by the present invention has a greater granulation tissue thickness, and it promotes the generation of more collagen fibers during wound healing. In short, the preparation of the present invention can better promote the repair of full-thickness skin wounds.

[0099] Figure 7 It is the network structure diagram of the hydrogel prepared by the present invention. Its typical feature is that the amino-modified end-hydroxyl triblock amphiphilic copolymer self-assembles into micelles in aqueous solution. The catechol groups on the CPD molecular chain react with the amino groups on the amino-modified end-hydroxyl triblock amphiphilic copolymer micelles through Schiff base or Michael addition under the oxidation of hydrogen peroxide to form a polymer network. At the same time, above its lowest dissolution temperature (such as under the condition of 25°C), the micelles assembled by the amino-modified end-hydroxyl triblock amphiphilic copolymer will undergo physical cross-linking through hydrophobic interactions or hydrogen bonds, further enhancing the cross-linking density. In addition, due to the presence of polymer micelles, hydrophobic drugs such as astragaloside can be further loaded in the micelles to play the function of drug delivery.

[0100] Example 7

[0101] Different from Example 1, the end-hydroxyl triblock amphiphilic polymer used in this example is Pluronic F68, and the prepared hydrogel dressing is named CPD / PF69-NH2@H2O25.

[0102] Example 8

[0103] Different from Example 1, the hydroxy-terminated triblock amphiphilic polymer used in this example is Pluronic F188, and the prepared hydrogel dressing is named CPD / PF188-NH2@H2O25.

[0104] Example 9

[0105] Different from Example 1, the hydroxy-terminated triblock amphiphilic polymer used in this example is poly(ethylene glycol)-b-poly(lactic acid)-b-poly(ethylene glycol), and the prepared hydrogel dressing is named CPD / (PEG-PLA-PEG)-NH2@H2O25.

[0106] Example 10

[0107] Different from Example 1, the hydroxy-terminated triblock amphiphilic polymer used in this example is poly(ethylene glycol)-b-poly(ε-caprolactone)-b-poly(ethylene glycol), and the prepared hydrogel dressing is named CPD / (PEG-PCL-PEG)-NH2@H2O25.

[0108] The present invention discloses an injectable antibacterial hemostatic hydrogel dressing with high strength and tissue adhesion strength, and its preparation method and application. First, a polymer poly(polyethylene glycol, citric acid) ester-g-dopamine (CPD) rich in catechol groups was designed and synthesized, where dopamine can provide ideal tissue adhesion strength. At the same time, an amino-functionalized Pluronic F127 (PF127-NH2) with amphiphilic segments was introduced into the polymer network to form micelles, which not only play a cross-linking role but also enhance the mechanical strength of the hydrogel dressing based on the mechanical dissipation mechanism. The polymer raw materials such as polyethylene glycol and Pluronic F127 (PF127) and the oxidation cross-linking system of hydrogen peroxide (H2O2) / horseradish peroxidase (HRP) used in the present invention have excellent biocompatibility, enabling it to have good application prospects as a hydrogel dressing in full-thickness wound repair, hemostasis, antibacterial, and drug delivery.

[0109] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of an injectable antibacterial hemostatic hydrogel dressing, characterized in that, Mix the CPD aqueous solution and the aqueous solution of the amino-modified end-hydroxyl triblock amphiphilic polymer to form a mixed system. Adjust the pH value of the mixed system to 6-8, then add hydrogen peroxide and the aqueous solution of horseradish peroxidase. After oxidative crosslinking, a polymer network is formed, which is a hydrogel dressing. The preparation process of CPD in the CPD aqueous solution is as follows: Mix citric acid, polyethylene glycol, and dopamine hydrochloride in a reaction vessel to form a first reaction system; under a nitrogen atmosphere, stir the first reaction system at a first set temperature to make the first reaction system in a molten state; lower the temperature of the molten first reaction system to a second set temperature, and stir the molten first reaction system at the second set temperature to react to obtain a reaction product. After dialyzing the reaction product, rotary evaporate and freeze-dry the dialyzed product to obtain CPD. The preparation process of the amino-modified end-hydroxyl triblock amphiphilic polymer in the aqueous solution of the amino-modified end-hydroxyl triblock amphiphilic polymer is as follows: Heat and melt the end-hydroxyl triblock amphiphilic polymer, dry the molten end-hydroxyl triblock amphiphilic polymer, and dissolve the dried end-hydroxyl triblock amphiphilic polymer in dichloromethane to form a second reaction system; add N,N'-carbonyldiimidazole to dichloromethane to form a third reaction system; drop the third reaction system into the second reaction system to form a fourth reaction system. Add ethylenediamine to the fourth reaction system, stir and then add water to form a fifth reaction system. After extracting the fifth reaction system with dichloromethane, wash the extraction product. Dissolve the washed precipitate in an organic solvent, centrifuge and separate, and then dry the centrifuged product to obtain the amino-modified end-hydroxyl triblock amphiphilic polymer.

2. The preparation method of an injectable antibacterial hemostatic hydrogel dressing according to claim 1, characterized in that, The concentration of the CPD aqueous solution is 500 mg / mL, and the concentration of the aqueous solution of the amino-modified end-hydroxyl triblock amphiphilic polymer is 300 mg / mL.

3. The preparation method of an injectable antibacterial hemostatic hydrogel dressing according to claim 1, characterized in that, Add 5-25 μL of hydrogen peroxide to 1 mL of the mixed system, and add 40 μL of the aqueous solution of horseradish peroxidase to 1 mL of the mixed system.

4. The preparation method of an injectable antibacterial hemostatic hydrogel dressing according to claim 1, characterized in that, Both the CPD aqueous solution and the aqueous solution of the amino-modified end-hydroxyl triblock amphiphilic polymer are pre-cooled and then mixed to form a mixed system.

5. The preparation method of an injectable antibacterial hemostatic hydrogel dressing according to claim 1, characterized in that, The mixed molar ratio of citric acid, polyethylene glycol, and dopamine hydrochloride is 1.1:1:0.5, the first set temperature is 160 °C, and the second set temperature is 140 °C.

6. The preparation method of an injectable antibacterial hemostatic hydrogel dressing according to claim 1, wherein Stir the molten first reaction system at the second set temperature for 48 h.

7. The preparation method of an injectable antibacterial hemostatic hydrogel dressing according to claim 1, characterized in that, The end-hydroxyl triblock amphiphilic polymer is Pluronic F68, Pluronic F127, Pluronic F188, polyethylene glycol-b-poly(lactic acid)-b-polyethylene glycol, or polyethylene glycol-b-poly(ε-caprolactone)-b-polyethylene glycol.

8. The preparation method of an injectable antibacterial hemostatic hydrogel dressing according to claim 1, characterized in that, The preparation process of the amino-modified end-hydroxyl triblock amphiphilic polymer is as follows: (2A) Heat and stir the hydroxyl-terminated triblock amphiphilic polymer in a nitrogen stream at 80 °C until it melts. Cut off the nitrogen stream and start vacuum pumping for 12 hours to dry the molten hydroxyl-terminated triblock amphiphilic polymer. (2B) Dissolve the dried hydroxyl-terminated triblock amphiphilic polymer in dichloromethane under an ice-water bath condition in a nitrogen stream to form a second reaction system. The temperature of the ice-water bath is less than 4 °C, and the concentration of the second reaction system is 50 mM. (2C) Dissolve N,N'-carbonyldiimidazole in dichloromethane to form a third reaction system. The concentration of the third reaction system is 800 mM. Add the third reaction system dropwise to the second reaction system under a nitrogen stream to form a fourth reaction system. The mixing volume ratio of the third reaction system to the second reaction system is 5:

4. (2D) Dropwise add ethylenediamine to the fourth reaction system under a nitrogen stream and stir to mix evenly to form a fifth reaction system. The molar ratio of ethylenediamine to the hydroxyl-terminated triblock amphiphilic polymer is 100:

1. (2E) Add deionized water to the fifth reaction system, extract with dichloromethane, and wash with saturated brine. Pre-cool the precipitate obtained from the washing in ether at -80 °C, and centrifuge to separate the precipitant and the product at a rotation speed of 8000 rpm. After drying, the final product can be obtained.

9. An injectable antibacterial hemostatic hydrogel dressing prepared by the preparation method according to any one of claims 1-8, characterized in that, It includes an amino-modified hydroxyl-terminated triblock amphiphilic copolymer and CPD; the amino-modified hydroxyl-terminated triblock amphiphilic copolymers are crosslinked with each other, and the amino-modified hydroxyl-terminated triblock amphiphilic copolymer and CPD are crosslinked through Schiff base or Michael addition.

10. Use of the injectable antibacterial hemostatic hydrogel dressing according to claim 9, characterized in that, The hydrogel adjuvant is used in full-thickness skin wound repair, hemostasis, antibacterial, or drug carriers.

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