WO2023197700A1 - 一种伤口敷料的制备方法、伤口敷料及应用 - Google Patents

一种伤口敷料的制备方法、伤口敷料及应用 Download PDF

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WO2023197700A1
WO2023197700A1 PCT/CN2023/070596 CN2023070596W WO2023197700A1 WO 2023197700 A1 WO2023197700 A1 WO 2023197700A1 CN 2023070596 W CN2023070596 W CN 2023070596W WO 2023197700 A1 WO2023197700 A1 WO 2023197700A1
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pdp
hydrogel
wound dressing
polymer
pdpc
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PCT/CN2023/070596
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French (fr)
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陈陶
胡杉杉
杨梓心
季平
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重庆医科大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0009—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials
    • A61L26/0014—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing macromolecular materials obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0004—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form containing inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0066—Medicaments; Biocides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/008—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L26/00—Chemical aspects of, or use of materials for, wound dressings or bandages in liquid, gel or powder form
    • A61L26/0061—Use of materials characterised by their function or physical properties
    • A61L26/0085—Porous materials, e.g. foams or sponges
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F8/00—Chemical modification by after-treatment
    • C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
    • C08F8/32—Introducing nitrogen atoms or nitrogen-containing groups by reaction with amines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/404—Biocides, antimicrobial agents, antiseptic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/04—Materials for stopping bleeding
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions

Definitions

  • the present disclosure relates to the technical field of medical materials, and specifically, to a preparation method of a wound dressing, a wound dressing and its application.
  • Tissue trauma is one of the most common clinical diseases. In the past decade, the cost of promoting wound care and healing has continued to increase. According to statistics, approximately 5.8 million people worldwide die from severe trauma every year. The first step in wound healing is to stop bleeding. Excessive blood loss after severe trauma can easily cause hypotension, multiple organ failure, and even death. Therefore, rapid control of bleeding is of great importance. In addition, exposed wounds can easily be infected by bacteria, causing difficult-to-heal wounds, seriously affecting human health and even causing death.
  • tissue adhesives, sealants, hemostatic agents, etc. have been widely used in wound closure, hemostasis, healing and other fields to treat severe wounds, incisions, infections and related complications.
  • fibrin glue, albumin-glutaraldehyde bioglue, cyanoacrylate, etc. have been widely used in clinical practice.
  • Fibrin glue has insufficient bonding strength, limited sources, high cost, and can easily cause potential infection and immunogenicity.
  • albumin-glutaraldehyde bioglue and cyanoacrylate adhesives have strong tissue bonding strength, their potential toxicity limits their application, and their bonding effect on wet tissues is limited.
  • tissue adhesives or hemostatic agents have difficulty preventing subsequent potential wound infection.
  • wound dressings loaded with antibacterial agents such as antibiotics, metal ions and quaternary ammonium salts, are often used to promote wound healing.
  • antibacterial agents such as antibiotics, metal ions and quaternary ammonium salts
  • traditional antibacterial agents are often cytotoxic and can induce the development of drug-resistant bacteria.
  • the present disclosure provides a method for preparing a wound dressing, which includes: reacting polyvinyl alcohol with 3,4-dihydroxyphenylalanine to obtain a PDP polymer, and complexing the PDP polymer with copper ions to form a hydrogel.
  • the method includes:
  • the PDP polymer is redissolved and then mixed and reacted with a copper salt to complex the PDP polymer with copper ions.
  • the molar ratio of the polyvinyl alcohol to the 3,4-dihydroxyphenylalanine is 2.5-4.5:1.
  • the molar ratio of the polyvinyl alcohol to the 3,4-dihydroxyphenylalanine is 3-4:1.
  • the molecular weight of the polyvinyl alcohol is 85-124kDa.
  • the catalyst is NaHSO 4 ⁇ H 2 O.
  • the polyvinyl alcohol is dissolved in an organic solvent at a temperature of 90-110°C, and the organic solvent is dimethyl sulfoxide.
  • reaction time of the polyvinyl alcohol and the 3,4-dihydroxyphenylalanine is 20-24h, and the reaction temperature is 75-80°C.
  • reaction process is carried out in the presence of protective gas.
  • the post-treatment process includes: dialyzing, rotary evaporating and drying the reacted solution.
  • the dialysis time is 2-4h.
  • the drying is performed by freeze-drying, and the freeze-drying time is 1-2 days.
  • the PDP polymer is mixed and dissolved with water under heating conditions to obtain a PDP solution, and a copper salt solution is added dropwise to the PDP solution and reacted for 30-60 minutes.
  • the concentration of PDP polymer in the PDP solution is controlled to be 80-120 mg/mL.
  • the copper salt is selected from at least one of copper chloride, copper sulfate and copper nitrate.
  • the copper salt is CuCl 2 ⁇ 2H 2 O, and the mass ratio of PDP polymer to copper salt is 100:1-2.
  • the present disclosure also provides a wound dressing prepared by any one of the aforementioned preparation methods.
  • the present disclosure also provides the use of the aforementioned wound dressing in preparing tissue wound repair materials.
  • the present disclosure also provides a wound dressing for use in repairing tissue wounds.
  • the present disclosure also provides a method of treating trauma in a subject, including:
  • the wound dressing is administered to the subject in need thereof.
  • the subject suffers from surface trauma, venous injury, arterial injury or undergoes surgery.
  • the surgery is surgery related to the heart, liver, spleen or kidneys.
  • Figure 1 is a schematic diagram of the preparation process and effect of the wound dressing provided by the embodiment of the present disclosure
  • Figure 2 shows the SEM morphology and mapping pictures of PDP and PDPC hydrogels
  • Figure 3 shows the UV spectra of PVA and PDP
  • Figure 4 is the XPS image of PDPC hydrogel
  • Figure 5 shows the changes in the storage modulus (G′) and loss modulus (G′′) of PDPC in the strain range of 0-1000 of PDPC hydrogel;
  • Figure 6 shows the change curve of PDPC viscosity with shear rate
  • Figure 7 shows an example of PDPC hydrogel bonding to mouse organs
  • Figure 8 shows the bonding strength of PDP and PDPC hydrogels to pig skin
  • Figure 9 is a schematic diagram (see A in the figure), color change diagram (see B in the figure) and quantitative results of using DPPH to detect oxygen free radicals scavenging by different hydrogels;
  • Figure 10 is a schematic diagram (see A in the figure), color change diagram (see B in the figure) and quantitative results of using NBT to detect superoxide anion (O 2 ⁇ - ) scavenging by different hydrogels;
  • Figure 11 is a schematic diagram (see A in the figure), color change diagram (see B in the figure) and quantitative results of using TMB to detect hydroxyl radical (OH ⁇ ) scavenging by different hydrogels;
  • Figure 12 shows the detection and quantitative analysis of the ability of different hydrogels to improve oxidative stress in L929 cells using active oxygen probing DCFH-DA;
  • Figure 13 shows the detection and quantitative analysis of the oxygen generation ability of L929 cells using different hydrogels using oxygen probe Ru(dpp) 3 Cl 2 ;
  • Figure 14 shows the cell activity results after co-culture of different hydrogels with different concentrations and L929 cells for 1 day (see A in the figure), 3 days (see B in the figure) and 7 days (see C in the figure);
  • Figure 15 shows the heat map (see A in the figure) and temperature quantitative results (see B in the figure) of different hydrogels irradiated with a near-infrared photothermal instrument within 6 minutes;
  • Figure 16 shows the temperature changes of four photothermal cycles when irradiating different hydrogels with a near-infrared photothermal instrument
  • Figure 17 is a diagram of colonies after co-culture of different hydrogels with Escherichia coli (E.C) and Staphylococcus aureus (S.A) for 24 hours;
  • Figure 18 shows the live and dead bacterial staining images and quantitative results of different hydrogels co-cultured with Escherichia coli (E.C) and Staphylococcus aureus (S.A) for 24 hours;
  • Figure 19 shows the crystal violet staining and quantitative results of different hydrogels co-cultured with Escherichia coli (E.C) and Staphylococcus aureus (S.A) bacterial biofilms for 24 hours;
  • Figure 20 shows the fluorescence staining images of different hydrogels after being co-cultured with Escherichia coli (E.C) and Staphylococcus aureus (S.A) bacterial biofilms for 48 hours;
  • E.C Escherichia coli
  • S.A Staphylococcus aureus
  • Figure 21 is a diagram of colonies after different hydrogels were co-cultured with Escherichia coli (E.C) and Staphylococcus aureus (S.A) for 24 hours and irradiated with near-infrared light for 5 minutes;
  • E.C Escherichia coli
  • S.A Staphylococcus aureus
  • Figure 22 shows the live and dead staining of bacteria and their quantitative results after different hydrogels were co-cultured with Escherichia coli (E.C) and Staphylococcus aureus (S.A) for 24 hours and irradiated with near-infrared light for 5 minutes;
  • E.C Escherichia coli
  • S.A Staphylococcus aureus
  • Figure 23 shows the crystal violet staining and quantitative results of different hydrogels co-cultured with Escherichia coli (E.C) and Staphylococcus aureus (S.A) bacterial biofilms for 24 hours and then irradiated with near-infrared light for 5 minutes;
  • E.C Escherichia coli
  • S.A Staphylococcus aureus
  • Figure 24 shows the fluorescence staining images of different hydrogels co-cultured with Escherichia coli (E.C) and Staphylococcus aureus (S.A) bacterial biofilms for 48 hours and then irradiated with near-infrared light for 5 minutes;
  • E.C Escherichia coli
  • S.A Staphylococcus aureus
  • Figure 25 is a diagram showing PDPC hydrogel’s effect on liver hemostasis of SD rats (see A in the diagram) and quantitative diagram of bleeding volume (see B in the diagram);
  • Figure 26 is a diagram showing the process of PDPC hydrogel hemostasis on the heart of SD rats.
  • Figure 27 is a diagram showing the process of PDPC hydrogel hemostasis in the carotid artery of New Zealand white rabbits;
  • Figure 28 is a diagram showing the hemostatic process of PDPC hydrogel on linear bleeding wounds in the liver of Bama mini pigs;
  • Figure 29 is a diagram showing the hemostatic process of PDPC hydrogel on round bleeding wounds in the liver of Bama mini pigs;
  • Figure 30 is a diagram showing the process of PDPC hydrogel hemostasis in the heart of Bama mini pigs
  • Figure 31 is a diagram showing the process of PDPC hydrogel hemostasis in the carotid artery of Bama mini pigs
  • Figure 32 shows the characterization of the wound healing status of PDPC hydrogel on the back skin of BALBc mice.
  • A is a diagram of the wound healing process of PDPC hydrogel on the back skin of BALBc mice;
  • B is a verification of the hydrogel through staining. Promote the formation of wound collagen fibers, promote angiogenesis and reduce inflammatory response to accelerate the wound healing process;
  • Figure 33 is a diagram of bacterial colonies extracted after different groups of hydrogels treated the back skin wounds of BALBc mice;
  • Figure 34 shows the blood routine and blood biochemical analysis results after 10 days of PDPC hydrogel treatment of back skin wounds in BALBc mice;
  • Figure 35 shows the results of hemolysis experiments of different PDPC hydrogels
  • Figure 36 is a picture of the PDP solution mixed with CuCl 2 ⁇ 2H 2 O and CuSO 4 respectively.
  • the embodiment of the present disclosure provides a method for preparing a wound dressing, which uses polyvinyl alcohol and 3,4-dihydroxyphenylalanine to react to obtain a PDP polymer, and combines the PDP polymer with copper The ions complex to form a hydrogel.
  • the prepared hydrogel integrates multiple functions such as tissue adhesion, rapid hemostasis, ROS scavenging activity, near-infrared photothermal responsiveness, and antibacterial properties, realizing wound healing. It has multiple effects of rapid surface hemostasis and anti-inflammatory and antibacterial effects.
  • a dehydration condensation reaction is carried out with 3,4-dihydroxyphenylalanine in the presence of a catalyst. After the reaction is completed, a PDP polymer in a gel state is obtained through post-treatment.
  • the hydroxyl group on polyvinyl alcohol and the carboxyl group on 3,4-dihydroxyphenylalanine are used for dehydration condensation, so that 3,4-dihydroxyphenylalanine is grafted onto the long chain of polyvinyl alcohol.
  • the molar ratio of polyvinyl alcohol to 3,4-dihydroxyphenylalanine is 2.5-4.5:1.
  • the molar ratio of polyvinyl alcohol to 3,4-dihydroxyphenylalanine is 3-4:1.
  • the polyvinyl alcohol has a molecular weight of 85-124kDa, such as 90-120kDa, 95-115kDa or 100-110kDa, such as 85kDa, 90kDa, 95kDa, 100kDa, 105kDa, 110kDa, 115kDa, 120kDa, 123kDa or 124kDa, or any The interval value between two endpoint values.
  • the molar ratio of polyvinyl alcohol to 3,4-dihydroxyphenylalanine can be 2.5:1, 2.8:1, 3.0:1, 3.2:1, 3.4:1, 3.5:1, 3.8:1 , 4.0:1, 4.2:1, 4.4:1, 4.5:1, etc., or the interval value between any two endpoint values, or any value between the above adjacent molar ratio values.
  • the catalyst is NaHSO 4 ⁇ H 2 O.
  • the above raw materials are all suitable as reaction catalysts to promote the rapid grafting of 3,4-dihydroxyphenylalanine.
  • the catalyst can be one type or several types.
  • polyvinyl alcohol is dissolved in an organic solvent at a temperature of 90-110°C, and the organic solvent is dimethyl sulfoxide.
  • the amount of organic solvent is not limited, as long as the polyvinyl alcohol can be fully dissolved to facilitate the reaction.
  • the reaction time of polyvinyl alcohol and 3,4-dihydroxyphenylalanine is 20-24h, and the reaction temperature is 75-80°C.
  • 3,4-dihydroxyphenylalanine can be increased. Grafting rate of hydroxyphenylalanine.
  • the reaction time can be 20h, 21h, 22h, 23h, 24h, etc., or an interval value between any two endpoint values, or any value between the above adjacent time values.
  • the reaction temperature can be 75°C, 76°C, 77°C, 78°C, 79°C, 80°C, etc., or an interval value between any two endpoint values, or between the above adjacent temperature values. Any value.
  • the reaction process is carried out in the presence of protective gas to prevent oxidation.
  • protective gas is not limited and can be nitrogen or other inert gases.
  • the inert gas includes helium, neon, argon, krypton, or xenon.
  • the post-treatment process includes: dialyzing, rotary evaporating and drying the reacted solution; the dialysis time is 2-4h, such as 2h, 2.2h, 2.5h, 2.8h, 3.0h, 3.2h, 3.5h, 3.8h, 4h, or the interval value between any two endpoint values; drying is done by freeze-drying, and the freeze-drying time is 1-2 days.
  • the reacted solution is put into a dialysis bag for processing to achieve the purpose of purifying the PDP polymer. The excess water and solvent are removed by rotary evaporation and then freeze-dried to obtain an intermediate product in a gel state.
  • the PDP polymer in the gel state is redissolved and then mixed and reacted with the copper salt.
  • the present disclosure utilizes the effects of hydrogen bonding and copper ion metal coordination to complex the PDP polymer with copper ions to form a hydrogel, which has good flexibility and injectability.
  • Cu + and Cu 2+ gives it strong antioxidant activity, and Cu ions also have excellent near-infrared photothermal response performance and antibacterial effect.
  • Cu also has the advantages of low price, simple synthesis, and good biocompatibility.
  • the PDP polymer is mixed and dissolved with water under heating conditions to obtain a PDP solution.
  • the copper salt solution is added dropwise to the PDP solution and reacts for 30-60min, such as 35-55min, 40-50min or 42-48min. , such as 30min, 35min, 40min, 45min, 50min, 55min or 60min, or an interval value between any two endpoint values.
  • the freeze-dried PDP polymer can be dissolved in water under heating conditions in a water bath at 60° C., and then a copper salt solution can be added dropwise for complexation to form a hydrogel.
  • the concentration of the PDP polymer in the PDP solution is controlled to be 80-120 mg/mL. Under this concentration condition, it is easy to complex with copper ions to form a hydrogel state.
  • the PDP polymer concentration is, for example, 84-116 mg/mL, 92-110 mg/mL or 95-105 mg/mL, such as 80 mg/mL, 85 mg/mL, 90 mg/mL, 95 mg/mL, 100 mg/mL, 105 mg /mL, 110mg/mL, 115mg/mL, 120mg/mL, etc., or an interval value between any two endpoint values, or any value between the above adjacent concentration values.
  • the copper salt is selected from at least one of copper chloride, copper sulfate, and copper nitrate.
  • the copper salt is CuCl 2 ⁇ 2H 2 O.
  • the mass ratio of PDP polymer to copper salt is 100:1-2 (such as 100:1, 100:1.1, 100:1.3, 100:1.5, 100:1.7, 100:1.9 or 100:2, etc., or the interval value between any two endpoint values).
  • the concentration of the copper salt solution is larger and can be controlled to 100-200mg/mL, such as 110-190mg/mL, 125-175mg/mL or 135-165mg/mL, such as 100mg/mL, 110mg/mL, 120mg/mL, 130mg /mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL, 200mg/mL, or an interval value between any two endpoint values, so that the complexation can form Hydrogel state.
  • One embodiment of the present disclosure also provides a wound dressing prepared by the above preparation method.
  • FIG. 1 is a schematic diagram of the synthesis strategy of PDPC hydrogel
  • FIG. 2 is a schematic diagram of PDPC hydrogel used for hemostasis in the carotid arteries, heart and liver of different animals (rats, rabbits, pigs)
  • the wound dressing provided by the embodiment of the present disclosure can not only quickly promote hemostasis in the wound area in the early stage of trauma, but also can effectively resist antibacterial and anti-infection, significantly promote acute (trauma and surgery) and chronic infected wounds (such as wounds of diabetic patients), and is expected to be effective Promote wound healing of various wounds.
  • the embodiment of the present disclosure also provides the application of the above wound dressing in preparing tissue wound repair materials.
  • the wound dressing provided by the embodiment of the present disclosure can be used as a basis and combined with other raw materials to prepare materials for tissue wound repair.
  • One embodiment of the present disclosure also provides a wound dressing for use in repairing tissue wounds.
  • One embodiment of the present disclosure also provides a method for treating trauma in a subject, including:
  • the wound dressing is administered to the subject in need thereof.
  • the subject suffers from surface trauma, venous injury, arterial injury, or undergoes surgery.
  • the surgery is surgery related to the heart, liver, spleen, or kidneys.
  • the present disclosure provides a wound dressing and a preparation method thereof.
  • the wound dressing has a rapid hemostatic effect on the wound surface and can simultaneously protect the wound surface and exert anti-inflammatory and antibacterial effects.
  • a PDP polymer is obtained by performing a dehydration condensation reaction between polyvinyl alcohol and 3,4-dihydroxyphenylalanine, and the PDP polymer is complexed with copper ions to form a hydrogel.
  • the prepared wound dressing has a rich porous structure and can stop bleeding by quickly absorbing blood. It is a multifunctional dressing that combines tissue adhesion, rapid hemostasis, ROS scavenging activity, near-infrared photothermal responsiveness, and antibacterial properties.
  • the all-in-one material can firmly adhere to the wound surface to quickly stop bleeding, protect the wound, exert anti-inflammatory effects, exert efficient antibacterial effects, and quickly promote wound healing, and has broad clinical application prospects.
  • This embodiment provides a method for preparing a wound dressing, which includes the following steps:
  • PDP polymer Dissolve 75 mmol of polyvinyl alcohol (PVA) with a molecular weight of 85-124 kDa in 180 mL of DMSO at 100°C. After dissolution, add 11 g of NaHSO 4 ⁇ H 2 O for catalysis. Wait until When the temperature dropped to 78°C, 20 mmol of 3,4-dihydroxyphenylalanine (DOPA) was added. React for 22 hours under N2 protection. After the reaction is completed, when the temperature drops to room temperature, the resulting solution is dialyzed in a dialysis bag for 3 days. The excess water is removed with a suspension evaporator, and then freeze-dried with a freeze dryer for 2 days to obtain dry PDP polymerization. things.
  • PVA polyvinyl alcohol
  • DOPA 3,4-dihydroxyphenylalanine
  • Example 2 The only difference from Example 1 is that the mass ratio of PDP polymer and CuCl 2 ⁇ 2H 2 O is controlled to 100:2, expressed as PDPC2.
  • This embodiment provides a method for preparing a wound dressing, which includes the following steps:
  • This embodiment provides a method for preparing a wound dressing, which includes the following steps:
  • PDP polymer Dissolve 80 mmol of polyvinyl alcohol (PVA) with a molecular weight of 13-23 kDa in 200 mL of DMSO at 110°C. After dissolution, add 12 g of NaHSO 4 ⁇ H 2 O for catalysis. Wait until When the temperature dropped to 80°C, 24 mmol of 3,4-dihydroxyphenylalanine (DOPA) was added. React for 24 hours under N2 protection. After the reaction is completed, when the temperature drops to room temperature, the resulting solution is dialyzed in a dialysis bag for 4 days. The excess water is removed with a suspension evaporator, and then freeze-dried with a freeze dryer for 2 days to obtain dry PDP polymerization. things.
  • PVA polyvinyl alcohol
  • DOPA 3,4-dihydroxyphenylalanine
  • Example 1 The only difference from Example 1 is that the mass ratio of PDP polymer and CuCl 2 ⁇ 2H 2 O is controlled to 100:3, represented by PDPC3.
  • Example 1 The only difference from Example 1 is that CuCl 2 ⁇ 2H 2 O is replaced by CuSO 4 of equal mass.
  • the PDP polymer and PDPC1 and PDPC2 hydrogels exhibit a uniform porous structure, and Cu ions are evenly distributed in the PDPC1 and PDPC2 hydrogels.
  • PDP polymer has an obvious characteristic peak at around 280nm. This characteristic peak is the characteristic peak of the catechol structure, proving that DOPA is successfully grafted onto the PVA polymer chain. This proves the successful synthesis of PDP polymer.
  • XPS not only proves that the PDPC hydrogel contains copper ions, but also proves the successful introduction of copper ions into the PDPC hydrogel.
  • Figure 7 shows examples of adhesion of PDPC hydrogel to mouse kidney, lung and liver respectively. As can be seen from Figure 7, the PDPC hydrogel showed good adhesion properties to various parts of the mouse.
  • control group used in this test example is a blank control without any treatment
  • Example 1 Use DPPH (MCE) as the ROS indicator. Add the DPPH prepared in advance with absolute ethanol into the EP tube, then add hydrogels of different components into groups, and react in the dark at room temperature for 10 minutes.
  • Test Example 1 and Implementation The results of the oxygen free radical scavenging ability of the PDP, PDPC1 and PDPC2 hydrogels prepared in Example 2 are shown in Figure 9.
  • A represents the principle diagram of scavenging oxygen free radicals
  • B represents the color change diagram of different hydrogels reacting with DPPH for 10 minutes
  • C represents the quantitative results of scavenging oxygen free radicals after reacting different hydrogels with DPPH for 10 minutes.
  • the color change of DPPH was used for analysis.
  • the DPPH was reacted with the hydrogel for 10 minutes to record the color change, and quantitative analysis was performed with a microplate reader.
  • the PDPC hydrogel has a good ability to scavenge oxygen free radicals.
  • PDPC2 is better than PDPC1 hydrogel.
  • A represents the principle diagram of scavenging superoxide anions (O 2 ⁇ - );
  • B represents the color change diagram of different hydrogels reacting with NBT for 30 min;
  • C represents the scavenging of O 2 ⁇ after reacting different hydrogels with DPPH for 30 min.
  • Quantitative results
  • PDPC hydrogel has a good ability to scavenge superoxide anions.
  • PDPC2 hydrogel has a further improved ability to scavenge superoxide anions.
  • NBT (Sigma) is selected as the indicator of hydroxyl radicals. This substance can be oxidized by hydroxyl radicals to produce a blue product.
  • A represents the principle diagram of scavenging hydroxyl radicals (OH ⁇ );
  • B represents the color change diagram of different hydrogels reacting with TMB for 30 min, and
  • C represents the quantitative results of scavenging OH ⁇ after reacting different hydrogels with DPPH for 30 min. .
  • PDPC hydrogel has good ability to scavenge hydroxyl radicals.
  • PDPC2 hydrogel has further improved ability to scavenge hydroxyl radicals.
  • DCFH-DA was selected as the intracellular ROS indicator. It itself has no fluorescence, but can react with ROS to emit strong green fluorescence. Add H 2 O 2 and materials into the well plate in which L929 cells have been seeded, and incubate for 24 hours. Add DCFH-DA to test the oxidative response of the PDP, PDPC1 and PDPC2 hydrogels prepared in Example 1 and Example 2 to L929 cells. Stimulate improvement capabilities, the results are shown in Figure 12.
  • A represents the fluorescence staining diagram of using DCFH-DA probe to detect ROS in L929 cells under oxidative stress state using different hydrogels; B represents the corresponding fluorescence intensity.
  • PDPC hydrogel has better ability to resist oxidative stress. Compared with PDPC1 hydrogel, PDPC2 hydrogel has further improved ability to resist oxidative stress.
  • PDPC has better antioxidant properties and is proportional to the content of copper ions. That is, compared to PDPC1 hydrogel, the antioxidant properties of PDPC2 hydrogel are Performance is further improved.
  • control group used in this test example is a blank control without any treatment
  • Hydrogels with different components and concentrations were added to well plates inoculated with L929 cells and co-cultured at different time points, and the toxicity of the PDP, PDPC1 and PDPC2 hydrogels prepared in Example 1 and Example 2 to L929 cells was tested.
  • the results are shown in Figure 14.
  • A represents the cell activity after 1 day of co-culture of different hydrogels with different concentrations and L929 cells;
  • B represents the cell activity after 3 days of co-culture of different hydrogels with different concentrations and L929 cells;
  • C represents the cell activity of different concentrations of different water Cell viability after co-culture of gel and L929 cells for 7 days.
  • control group used in this test example is a blank control without any treatment
  • Figure 16 shows the temperature rise test of PDPC hydrogel under the same conditions for 4 cycles of laser irradiation.
  • the PDPC hydrogel showed good photothermal properties during the 4 different cycles, confirming that the PDPC hydrogel has excellent photostability. sex.
  • control group used in this test example is a blank control without any treatment
  • Figure 17 shows the colony diagram of different hydrogels co-cultured with Escherichia coli (E.C) (A) and Staphylococcus aureus (S.A) (B) for 24 hours, proving that PDPC hydrogel can inhibit the growth of both bacteria. colony formation;
  • Figure 18 shows the live and dead bacterial staining (Live/Dead double staining kit Sigma) pictures and quantitative results of different hydrogels co-cultured with E.C (A) and S.A (B) for 24 hours, proving that PDPC hydrogels Glue effectively kills two types of bacteria;
  • Figure 19 shows the crystal violet staining and quantitative results of different hydrogels co-cultured with E.C (A) and S.A (B) for 24 hours, proving that PDPC hydrogel can effectively kill bacterial biofilms;
  • Figure 20 shows the fluorescence staining images of different hydrogels co-cultured with E.C (A) and S.A (B) bacterial biofilms for 48 hours, which also proves that PDPC hydrogel can effectively kill bacterial biofilms.
  • control group used in this test example is a blank control without any treatment
  • Figure 21 shows the colony diagram of different hydrogels after they were co-cultured with Escherichia coli (E.C) (A) and Staphylococcus aureus (S.A) (B) for 24 hours and irradiated with 808nm near-infrared light for 5 minutes, proving that PDPC hydrogels After the glue is illuminated, the colony formation of the two bacteria can be further inhibited.
  • E.C Escherichia coli
  • S.A Staphylococcus aureus
  • Figure 22 shows the live and dead staining of bacteria and its quantitative results after different hydrogels were co-cultured with E.C (A) and S.A (B) for 24 hours and irradiated with 808nm near-infrared light for 5 minutes, proving that after PDPC hydrogel is illuminated Can kill two kinds of bacteria more effectively.
  • Figure 23 shows the crystal violet staining and quantitative results of different hydrogels co-cultured with E.C (A) and S.A (B) for 24 hours and irradiated with 808nm near-infrared light for 5 minutes, proving that PDPC hydrogels can be effective after irradiation Kill bacterial biofilms;
  • Figure 24 shows the fluorescence staining pictures of different hydrogels co-cultured with E.C (A) and S.A (B) bacterial biofilms for 48 hours and irradiated with 808nm near-infrared light for 5 minutes. It also proves that PDPC hydrogels can be effective after being illuminated Kills bacterial biofilms.
  • control group used in this test example is Hustar commercial gelatin sponge.
  • Figure 25 shows the quantitative results of PDPC hydrogel hemostasis in the liver of SD rats.
  • a in Figure 25 represents the bleeding situation of the liver within 60 s, and B represents the quantitative results of liver blood loss within 60 s.
  • the PDPC hydrogel has better hemostatic properties and can quickly stop bleeding in the liver of rats, and the blood loss is significantly reduced compared to the blank group;
  • Figure 26 shows the process of PDPC hydrogel hemostasis in the heart of SD rats. It can be seen from the figure that after applying PDPC2 hydrogel to the heart wound of the rat, the PDPC2 hydrogel can quickly and effectively seal the heart wound. Stop bleeding in the heart, proving its excellent hemostatic properties;
  • Figure 27 shows the process of PDPC hydrogel hemostasis in the carotid artery of New Zealand white rabbits.
  • the carotid artery is cut off and a wound is made, and then the wound is coated with PDPC2 hydrogel. After the artery clamp is loosened, the PDPC2 hydrogel condenses.
  • the glue can successfully seal the wound bleeding of the pig carotid artery, and the carotid artery can restore its blood supply after creating a new wound at the distal end of the original opening, indicating that the normal blood supply of the rabbit is not affected after the application of the material.
  • Figure 28 is a diagram of the hemostatic process of PDPC hydrogel on the linear bleeding wound of the liver of Bama mini pigs
  • Figure 29 is a diagram of the hemostatic process of PDPC hydrogel on the round bleeding wound of the liver of Bama mini pigs.
  • using PDPC2 hydrogel to cover linear and circular bleeding wounds in pig livers can quickly and successfully seal the wounds and achieve hemostasis. It has been proven that PDPC hydrogel can seal bleeding in large animals such as pigs and has excellent hemostatic properties.
  • FIG. 30 is a diagram of the process of PDPC hydrogel hemostasis on the heart of Bama mini pigs
  • Figure 31 is a diagram of the process of PDPC hydrogel on hemostasis of carotid arteries of Bama mini pigs.
  • PDPC hydrogel can not only quickly seal the bleeding in the heart and carotid arteries of small and medium-sized animals such as rats and rabbits, but also can seal the bleeding in the heart and carotid arteries of large animals such as pigs, even under large blood pressure in the heart and carotid arteries. It can still adhere firmly to the surface of wound tissue under impact, achieving a rapid hemostasis effect.
  • control group used in this test example is a blank control without any treatment
  • Figure 32 is a diagram of the wound healing process of PDPC hydrogel on the back skin of BALBc mice. H&E and Sirius red staining were used to verify that the hydrogel promoted the formation of collagen fibers in the wound. Fluorescent staining probes were further used to label ⁇ -SMA, CD31, iNOS and CD206, through Nikon, Ci-POL fluorescence microscopy observation, it was verified that PDPC hydrogel reduced wound inflammation and promoted wound angiogenesis. As shown in the figure, PDPC hydrogel healed faster than the wounds of mice in other groups, proving that PDPC hydrogel The gel effectively promotes skin wound healing.
  • Figure 33 is a colony diagram of bacterial fluid extracted from the wound after different groups of hydrogels treated mouse back skin wounds. As shown in the figure, PDPC hydrogel can significantly reduce the number of bacteria in the wound after treating mouse skin wounds. It is proved that PDPC hydrogel has good antibacterial properties, thereby promoting skin wound healing.
  • control group used in this test example is a blank control without any treatment
  • FIG 34 shows the white blood cells (WBC) (A), red blood cells (RBC) (B), platelets (PLT) (C), serum creatine kinase (CK) (D), and lactic acid detoxification after hydrogel treatment in different groups.
  • WBC white blood cells
  • RBC red blood cells
  • PPT platelets
  • CK serum creatine kinase
  • E Hydrogenase
  • E alanine aminotransferase
  • AST aspartate aminotransferase
  • G urea nitrogen
  • BUN urea nitrogen
  • CREA creatinine
  • the WBC, RBC and PLT of different groups of hydrogels after 10 days of wound treatment were not significantly different from the blank control group, proving that PDPC hydrogel has no obvious blood toxicity; as shown in D-I in Figure 34 It shows that there is no significant difference between the CK, LDH, ALT, AST, BUN and CREA of different groups of hydrogels after 10 days of wound treatment and the blank control group, proving that PDPC hydrogel has no obvious toxicity to the heart, liver, and kidneys. It is proved that PDPC hydrogel has excellent in vivo biocompatibility.
  • the colors of the PDP, PDPC1 and PDPC2 hydrogel groups are similar to the negative control group (PBS), indicating that no obvious hemolysis occurred, while the PDPC3 hydrogel group is similar to the positive control group (TritonX-100) , showing bright red color, indicating severe hemolysis.
  • PBS negative control group
  • TritonX-100 positive control group
  • the hemostatic effect of PDPC2 hydrogel is significantly better than that of gelatin sponge.
  • the bleeding volume is significantly less than that of gelatin sponge, and the difference is statistically significant, proving that PDPC hydrogel is more effective than commercial gelatin sponge. Achieve better hemostatic effect, reduce patient bleeding volume, and accelerate patient wound healing.
  • the PDPC after mixing the PDP solution and the CuCl 2 ⁇ 2H 2 O solution is in a uniformly distributed solution state, while the PDPC after mixing the PDP solution and the CuSO 4 solution has obvious precipitation, that is, CuSO 4
  • the solution cannot fully react with the PDP solution to form a uniform PDPC solution, nor can it form the required PDPC hydrogel. Therefore, this disclosure selects CuCl 2 ⁇ 2H 2 O to prepare PDPC hydrogel.
  • the present disclosure provides a wound dressing preparation method, wound dressing and application, using polyvinyl alcohol (PVA) as a carrier, integrating the excellent wet adhesive properties of DOPA and the excellent characteristics of metal ion Cu , achieving dynamic cross-linking of catechol groups and preparing a biomimetic mussel multifunctional hydrogel (PDPC hydrogel).
  • PVA polyvinyl alcohol
  • This hydrogel has the following excellent properties:
  • the present disclosure provides a wound dressing, which has a rich porous structure and can stop bleeding of the wound by quickly absorbing blood. It is a wound dressing that combines tissue adhesion, rapid hemostatic properties, ROS scavenging activity, near-infrared photothermal responsiveness, and antibacterial properties. It can firmly adhere to the wound surface to quickly stop bleeding, protect the wound, exert anti-inflammatory effects, exert efficient antibacterial effects, and quickly promote wound healing.
  • the preparation method disclosed is simple and easy to scale up. production, therefore the wound dressing and its preparation method of the present disclosure have broad clinical application prospects.

Abstract

本公开提供了一种伤口敷料的制备方法、伤口敷料及应用，涉及医用材料技术领域。伤口敷料的制备方法包括：利用聚乙烯醇与3,4-二羟基苯丙氨酸反应得到PDP聚合物，将PDP聚合物与铜离子络合形成PDPC水凝胶。制备得到的伤口敷料具有丰富的多孔结构，可通过快速吸收血液对伤口进行止血，是一种集组织粘附性、快速止血特性、ROS清除活性、近红外光热响应性、抗菌性等多功能于一体的材料，能够牢固粘附于伤口表面快速止血、保护创面并发挥抗炎效应，发挥高效的抗菌效果，快速促进创面伤口的愈合，具备广泛的临床应用前景。

Description

一种伤口敷料的制备方法、伤口敷料及应用
相关申请的交叉引用
本公开要求于2022年04月13日提交中国专利局的申请号为CN202210385179.1、名称为“一种伤口敷料的制备方法、伤口敷料及应用”的中国专利申请的优先权，其全部内容通过引用结合在本公开中。
技术领域
本公开涉及医用材料技术领域，具体而言，涉及一种伤口敷料的制备方法、伤口敷料及应用。
背景技术
组织创伤是临床最常见的疾病之一，在过去十年中，促进伤口护理和愈合的费用持续增加，据统计全球每年约有580万人死于严重创伤。伤口愈合的第一步是止血，严重创伤后导致的过度失血容易引起伤者低血压和多器官衰竭，甚至死亡。因此，迅速控制出血意义重大。此外，暴露的伤口很容易被细菌感染，造成难愈性伤口，严重影响人类健康，甚至造成死亡。
目前，组织粘接剂、密封剂、止血剂等已广泛应用于伤口闭合、止血、愈合等领域，用于治疗严重创伤、切口、感染及相关并发症。例如，纤维蛋白胶、白蛋白-戊二醛生物胶、氰基丙烯酸酯等，已广泛应用于临床。
但是，现在的几种材料在用于伤口治疗时均存在一定的问题。纤维蛋白胶粘接强度不足，且来源有限，成本高，易引起潜在的感染和免疫原性。而白蛋白-戊二醛生物胶和氰基丙烯酸酯粘接剂等虽然具备较强的组织粘接强度，然而其潜在的毒性限制了其应用，且对于湿润组织的粘接作用有限。
此外，这些组织粘接剂或止血剂难以防止后续潜在的伤口感染。为防止伤口感染，现阶段往往运用负载抗菌剂的伤口敷料促进伤口愈合，如抗生素、金属离子和季铵盐等。但传统的抗菌剂通常具有细胞毒性且能诱导耐药细菌的产生。
因此，开发具备良好组织粘接性能、抗菌、抗氧化应激、抗感染的止血粘接剂和伤口敷料具备重要的临床意义。
发明内容
本公开提供一种伤口敷料的制备方法，包括：利用聚乙烯醇与3,4-二羟基苯丙氨酸反应得到PDP聚合物，将PDP聚合物与铜离子络合形成水凝胶。
可选地，所述方法包括：
将聚乙烯醇溶解之后，在催化剂存在的条件下与3,4-二羟基苯丙氨酸反应，在反应完成之后通过后处理得到凝胶状态的PDP聚合物；
将PDP聚合物复溶之后与铜盐混合反应，以使PDP聚合物与铜离子络合。
可选地，所述聚乙烯醇与所述3,4-二羟基苯丙氨酸的摩尔比为2.5-4.5:1。
可选地，所述聚乙烯醇与所述3,4-二羟基苯丙氨酸的摩尔比为3-4:1。
可选地，所述聚乙烯醇的分子量为85-124kDa。
可选地，所述催化剂为NaHSO 4·H 2O。
可选地，将所述聚乙烯醇在90-110℃的条件下溶解于有机溶剂中，所述有机溶剂为二甲基亚砜。
可选地，所述聚乙烯醇和所述3,4-二羟基苯丙氨酸的反应时间为20-24h，反应温度为75-80℃。
可选地，反应过程在保护气存在的条件下进行。
可选地，后处理的过程包括：将反应后的溶液进行透析、旋蒸和干燥。
可选地，透析时间为2-4h。
可选地，所述干燥采用冻干的方式，冻干时间为1-2天。
可选地，将所述PDP聚合物在加热的条件下与水混合溶解得到PDP溶液，在所述PDP溶液中滴加铜盐溶液，反应30-60min。
可选地，控制所述PDP溶液中PDP聚合物浓度为80-120mg/mL。
可选地，所述铜盐选自氯化铜、硫酸铜和硝酸铜中的至少一种。
可选地，所述铜盐为CuCl 2·2H 2O，且PDP聚合物与铜盐的质量比为100:1-2。
本公开还提供一种伤口敷料，通过前述中任一项的制备方法制备而得。
本公开还提供前述伤口敷料在制备组织创伤修复材料中的应用。
本公开还提供一种伤口敷料，用于修复组织创伤的用途。
本公开还提供一种对受试者中创伤处理的方法，包括：
向所述有此需要的受试者施用所述的伤口敷料。
可选地，所述受试者患有体表创伤、静脉损伤、动脉损伤或经历了手术。
可选地，所述手术是与心脏、肝脏、脾脏或肾脏相关的手术。
附图说明
为了更清楚地说明本公开实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本公开的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本公开实施例提供的伤口敷料的制备流程和效果示意图；
图2为PDP和PDPC水凝胶的SEM形貌图和mapping图；
图3为PVA及PDP的紫外光谱图；
图4为PDPC水凝胶的XPS图像；
图5为PDPC水凝胶在0-1000应变范围内PDPC的储能模量(G′)和损耗模量(G″)的变化图；
图6为PDPC的黏度随剪切速率的变化曲线；
图7为PDPC水凝胶粘接小鼠脏器实例图；
图8为PDP和PDPC水凝胶对猪皮的粘接强度；
图9为利用DPPH检测不同水凝胶清除氧自由基的示意图(见图中A)、颜色变化图(见图中B)和定量结果；
图10为利用NBT检测不同水凝胶清除超氧阴离子(O 2· -)的示意图(见图中A)、颜色变化图(见图中B)和定量结果；
图11为利用TMB检测不同水凝胶清除羟基自由基(OH·)的示意图(见图中A)、颜色变化图(见图中B)和定量结果；
图12为利用活性氧探诊DCFH-DA检测不同水凝胶对L929细胞的氧化应激改善能力检测和定量分析；
图13为利用氧探针Ru(dpp) 3Cl 2检测不同水凝胶促使L929细胞的氧气生成能力检测和定量分析；
图14为不同浓度的不同水凝胶与L929细胞共培养1天(见图中A)、3天(见图中B)和7天(见图中C)后的细胞活性结果；
图15为利用近红外光热仪照射不同水凝胶6min内的热图(见图中A)和温度定量结果(见图中B)；
图16为利用近红外光热仪照射不同水凝胶4个光热循环的温度变化情况；
图17为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)共培养24h后的菌落图；
图18为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)共培养24h后的细菌活死染色图及其定量结果；
图19为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)细菌生物膜共培养24h后的结晶紫染色及定量结果图；
图20为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)细菌生物膜共培养48h后的荧光染色图；
图21为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)共培养24h后用近红外光照射5min后的菌落图；
图22为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)共培养24h后用近红外光照射5min后的细菌活死染色图及其定量结果；
图23为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)细菌生物膜共培养24h后用近红外光照射5min后的结晶紫染色及定量结果图；
图24为不同水凝胶分别与大肠杆菌(E.C)和金黄色葡萄球菌(S.A)细菌生物膜共培养48h后用近红外光照射 5min后的荧光染色图；
图25为PDPC水凝胶对SD大鼠肝脏止血图(见图中A)及出血量定量图(见图中B)；
图26为PDPC水凝胶对SD大鼠心脏止血过程图；
图27为PDPC水凝胶对新西兰大白兔颈动脉止血过程图；
图28为PDPC水凝胶对巴马小型猪的肝脏线性出血伤口的止血过程图；
图29为PDPC水凝胶对巴马小型猪的肝脏圆形出血伤口的止血过程图；
图30为PDPC水凝胶对巴马小型猪心脏止血过程图；
图31为PDPC水凝胶对巴马小型猪颈动脉止血过程图；
图32为PDPC水凝胶对BALBc小鼠背部皮肤伤口愈合状况的表征，其中，(A)为PDPC水凝胶对BALBc小鼠背部皮肤伤口愈合过程图；(B)为通过染色验证水凝胶促进创口胶原纤维形成、促进血管生成及减轻炎症反应以达到加快创口愈合的过程；
图33为不同组别水凝胶处理BALBc小鼠背部皮肤伤口后提取细菌的菌落图；
图34为PDPC水凝胶治疗BALBc小鼠背部皮肤伤口10天后的血常规和血生化分析结果；
图35为不同PDPC水凝胶的溶血实验结果图；
图36为PDP溶液分别与CuCl 2·2H 2O和CuSO 4混合后的图片。
具体实施方式
为使本公开实施方式和实施例的目的、技术方案和优点更加清楚，下面将对本公开实施方式和实施例中的技术方案进行清楚、完整地描述。实施方式和实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市售购买获得的常规产品。
请参照图1中(A)，本公开实施方式提供一种伤口敷料的制备方法，其利用聚乙烯醇与3,4-二羟基苯丙氨酸反应得到PDP聚合物，将PDP聚合物与铜离子络合形成水凝胶。参照图1中(B)和(C)，制备得到的水凝胶集组织粘附性、快速止血特性、ROS清除活性、近红外光热响应性、抗菌性等多功能于一体，实现了伤口表面快速止血和抗炎抗菌的多重效果。
本公开一实施方式所提供的伤口敷料制备方法如下：
S1、PDP聚合物的合成
将聚乙烯醇溶解之后，在催化剂存在的条件下与3,4-二羟基苯丙氨酸进行脱水缩合反应，在反应完成之后通过后处理得到凝胶状态的PDP聚合物。利用聚乙烯醇上的羟基和3,4-二羟基苯丙氨酸上的羧基进行脱水缩合，使3,4-二羟基苯丙氨酸接枝在聚乙烯醇的长链上。
在一些实施方式中，聚乙烯醇与3,4-二羟基苯丙氨酸的摩尔比为2.5-4.5:1。可选地，聚乙烯醇与3,4-二羟基苯丙氨酸的摩尔比为3-4:1。可选地，聚乙烯醇的分子量为85-124kDa，例如90-120kDa、95-115kDa或100-110kDa，诸如85kDa、90kDa、95kDa、100kDa、105kDa、110kDa、115kDa、120kDa、123kDa或124kDa，或者任意两个端点值之间的区间值。通过控制聚乙烯醇分子量以及两种原料的用量比，以提升产品的组织粘附性能以及止血效果。
可选地，聚乙烯醇与3,4-二羟基苯丙氨酸的摩尔比可以为2.5:1、2.8:1、3.0:1、3.2:1、3.4:1、3.5:1、3.8:1、4.0:1、4.2:1、4.4:1、4.5:1等，或者任意两个端点值之间的区间值，也可以为以上相邻摩尔比值之间的任意值。
在一些实施方式中，催化剂为NaHSO 4·H 2O。以上几种原料均适合于作为反应催化剂，促进3,4-二羟基苯丙氨酸的快速接枝，催化剂可以为一种，也可以为几种。
在一些实施方式中，将聚乙烯醇在90-110℃的条件下溶解于有机溶剂中，有机溶剂为二甲基亚砜。有机溶剂的用量不做限定，保证聚乙烯醇能够充分溶解便于进行反应即可。
在一些实施方式中，聚乙烯醇和3,4-二羟基苯丙氨酸的反应时间为20-24h，反应温度为75-80℃，通过进一步控制反应时间和反应温度以提升3,4-二羟基苯丙氨酸的接枝率。可选地，反应时间可以为20h、21h、22h、23h、24h等，或者任意两个端点值之间的区间值，也可以为以上相邻时间值之间的任意值。可选地，反应温度可以为75℃、76℃、77℃、78℃、79℃、80℃等，或者任意两个端点值之间的区间值，也可以为以上相邻温度值之间的任意值。
在一些实施方式中，反应过程在保护气存在的条件下进行，以防止氧化。可选地，保护气的种类不限，可以为氮气或其他惰性气体。可选地，惰性气体包括氦气、氖气、氩气、氪气或氙气。
在一些实施方式中，后处理的过程包括：将反应后的溶液进行透析、旋蒸和干燥；透析时间为2-4h，诸如2h、2.2h、2.5h、2.8h、3.0h、3.2h、3.5h、3.8h、4h，或者任意两个端点值之间的区间值；干燥采用冻干的方式，冻干时间为1-2天。可选地，透析是将反应后的溶液装入透析袋中进行处理，以达到使PDP聚合物得到纯化的目的，经过旋蒸去除多余水分和溶剂之后经过冻干得到凝胶状态的中间产物。
S2、PDPC水凝胶的合成
将凝胶状态的PDP聚合物复溶之后与铜盐混合反应。本公开利用氢键和铜离子金属配位的作用，以使PDP聚合物与铜离子络合形成水凝胶，该水凝胶具有较好的柔韧性和可注射性。
需要说明的是，Cu +和Cu 2+之间的可逆的电子转移赋予其强大的抗氧化活性，而且Cu离子还具备优异的近红外光热响应性能和抗菌效应。此外，Cu还具有价格便宜，合成简易、生物相容性良好的优点。
在实际操作过程中，将PDP聚合物在加热的条件下与水混合溶解得到PDP溶液，在PDP溶液中滴加铜盐溶液，反应30-60min，例如35-55min、40-50min或42-48min，诸如30min、35min、40min、45min、50min、55min或60min，或者任意两个端点值之间的区间值。可选地，例如可以将冻干的PDP聚合物在60℃水浴加热的条件下溶解于水中，再滴加铜盐溶液进行络合形成水凝胶。
在一些实施方式中，控制PDP溶液中PDP聚合物浓度为80-120mg/mL，在此浓度条件下便于和铜离子络合形成水凝胶状态。可选地，PDP聚合物浓度为例如84-116mg/mL、92-110mg/mL或95-105mg/mL，诸如80mg/mL、85mg/mL、90mg/mL、95mg/mL、100mg/mL、105mg/mL、110mg/mL、115mg/mL、120mg/mL等，或者任意两个端点值之间的区间值，也可以为以上相邻浓度值之间的任意值。
在一些实施方式中，铜盐选自氯化铜、硫酸铜和硝酸铜中的至少一种。可选地，铜盐为CuCl 2·2H 2O。可选地，PDP聚合物与铜盐的质量比为100:1-2(诸如100:1、100:1.1、100:1.3、100:1.5、100:1.7、100:1.9或100:2等，或者任意两个端点值之间的区间值)。铜盐溶液的浓度较大，可以控制为100-200mg/mL，例如110-190mg/mL、125-175mg/mL或135-165mg/mL，诸如100mg/mL、110mg/mL、120mg/mL、130mg/mL、140mg/mL、150mg/mL、160mg/mL、170mg/mL、180mg/mL、190mg/mL、200mg/mL，或者任意两个端点值之间的区间值，以使络合之后能够形成水凝胶状态。
本公开一实施方式还提供一种伤口敷料，通过上述制备方法制备而得。
需要说明的是，图1中(A)为PDPC水凝胶的合成策略示意图；(B)为PDPC水凝胶用于不同动物(大鼠、兔、猪)的颈动脉、心脏和肝脏止血示意图；(C)为PDPC水凝胶促进伤口愈合示意图。
本公开实施方式所提供的伤口敷料不仅能够在创伤早期快速促进伤口区域止血，且能够有效抗菌和抗感染，显著促进急性(外伤和手术)和慢性感染伤口(如糖尿病患者伤口)，可望有效促多种创伤的伤口愈合。
本公开实施方式还提供了上述伤口敷料在制备组织创伤修复材料中的应用，可以以本公开实施方式所提供的伤口敷料为基础，结合其他原料再制备形成用于组织创伤修复的材料。
本公开一实施方式还提供一种伤口敷料，用于修复组织创伤的用途。
本公开一实施方式还提供一种对受试者中创伤处理的方法，包括：
向所述有此需要的受试者施用所述的伤口敷料。
在一些实施方式中，受试者患有体表创伤、静脉损伤、动脉损伤或经历了手术。
在一些实施方式中，该手术是与心脏、肝脏、脾脏或肾脏相关的手术。
本公开提供了一种伤口敷料及其制备方法，该伤口敷料具有伤口表面快速止血效果，同时能够保护创面发挥抗炎、抗菌效果的伤口敷料。
本公开通过利用聚乙烯醇与3,4-二羟基苯丙氨酸进行脱水缩合反应得到PDP聚合物，将PDP聚合物与铜离子络合形成水凝胶。制备得到的伤口敷料具有丰富的多孔结构，可通过快速吸收血液对伤口进行止血，是一种集组织粘附性、快速止血特性、ROS清除活性、近红外光热响应性、抗菌性等多功能于一体的材料，能够牢固粘附于伤口表面快速止血、保护创面并发挥抗炎效应，发挥高效的抗菌效果，快速促进创面伤口的愈合，具备广泛的临床应用前景。
实施例
以下结合实施例对本公开的特征和性能作进一步的详细描述。
实施例1
本实施例提供一种伤口敷料的制备方法，包括如下步骤：
(1)PDP聚合物的合成：将75mmol、分子量为85-124kDa的聚乙烯醇(PVA)在100℃的条件下溶于180mL的DMSO中，溶解后加入11g NaHSO 4·H 2O催化，待温度降至78℃时，加入20mmol 3,4-二羟基苯丙氨酸(DOPA)。在N 2保护下反应22h，反应结束后待温度降至室温时将所得溶液在透析袋内透析3天，悬蒸仪去除多余水分，然后用冻干机冻干2天，得到干燥的PDP聚合物。
(2)PDPC水凝胶的合成：将冻干的PDP聚合物称重，在60℃水浴加热的条件下，按照100mg/mL的浓度溶解于去离子水中，再滴入氯化铜水溶液(采用CuCl 2·2H 2O溶于水制备而得)，氯化铜水溶液中铜离子浓度为100mg/mL，控制PDP聚合物和CuCl 2·2H 2O的质量比为100:1。充分搅拌反应30-60min，制备成PDPC水凝胶，用PDPC1表示。
实施例2
与实施例1的区别仅在于：控制PDP聚合物和CuCl 2·2H 2O的质量比为100:2，用PDPC2表示。
实施例3
本实施例提供一种伤口敷料的制备方法，包括如下步骤：
(1)PDP聚合物的合成：将70mmol、分子量为89-98kDa的聚乙烯醇(PVA)在90℃的条件下溶于150mL的DMSO中，溶解后加入10g NaHSO 4·H 2O催化，待温度降至75℃时，加入18mmol 3,4-二羟基苯丙氨酸(DOPA)。在N 2保护下反应20h，反应结束后待温度降至室温时将所得溶液在透析袋内透析2天，悬蒸仪去除多余水分，然后用冻干机冻干1天，得到干燥的PDP聚合物。
(2)PDPC水凝胶的合成：将冻干的PDP聚合物称重，在60℃水浴加热的条件下，按照80mg/mL的浓度溶解于去离子水中，再滴入氯化铜水溶液(采用CuCl 2·2H 2O溶于水制备而得)，氯化铜水溶液中铜离子浓度为200mg/mL，控制PDP聚合物和CuCl 2·2H 2O的质量比为100:2。充分搅拌反应30min，制备成PDPC水凝胶。
实施例4
本实施例提供一种伤口敷料的制备方法，包括如下步骤：
(1)PDP聚合物的合成：将80mmol、分子量为13-23kDa的聚乙烯醇(PVA)在110℃的条件下溶于200mL的DMSO中，溶解后加入12g NaHSO 4·H 2O催化，待温度降至80℃时，加入24mmol 3,4-二羟基苯丙氨酸(DOPA)。在N 2保护下反应24h，反应结束后待温度降至室温时将所得溶液在透析袋内透析4天，悬蒸仪去除多余水分，然后用冻干机冻干2天，得到干燥的PDP聚合物。
(2)PDPC水凝胶的合成：将冻干的PDP聚合物称重，在60℃水浴加热的条件下，按照120mg/mL的浓度溶解于去离子水中，再滴入氯化铜水溶液(采用CuCl 2·2H 2O溶于水制备而得)，氯化铜水溶液中铜离子浓度为150mg/mL，控制PDP聚合物和CuCl 2·2H 2O的质量比为100:2。充分搅拌反应60min，制备成PDPC水凝胶。
对比例1
与实施例1的区别仅在于：控制PDP聚合物和CuCl 2·2H 2O的质量比为100:3，用PDPC3表示。
对比例2
将常见市售的明胶海绵(沪士达)与本公开中PDPC水凝胶伤口敷料进行对比，比较二者对伤口止血的效果。
对比例3
与实施例1的区别仅在于：将CuCl 2·2H 2O替换为等质量的CuSO 4。
试验例1
对实施例中制备得到的PDPC水凝胶进行材料表征。
(1)采用扫描电子显微镜测试实施例1中得到的PDP聚合物，以及实施例1中制备得到的PDPC1和实施例2中制备得到的PDPC2水凝胶的SEM照片和mapping图，结果如图2所示。图2中A表示PDP聚合物的SEM照片，B表示PDPC1的SEM照片和mapping图，C表示PDPC2的SEM照片和mapping图。
从图2可以看出，PDP聚合物以及PDPC1和PDPC2水凝胶呈现出均匀的多孔结构，且PDPC1和PDPC2水凝胶中Cu离子均匀分布。
(2)测试实施例1中选用的PVA以及制备得到PDP聚合物的紫外光谱，结果如图3所示。
从图3可以看出相比PVA而言，PDP聚合物在280nm左右处具有明显的特征峰，该特征峰为邻苯二酚结构的特征峰，证明DOPA成功接枝到PVA聚合物链上，从而证明PDP聚合物的成功合成。
(3)采用X射线光电子能谱仪测试实施例1中得到PDPC1水凝胶的XPS图像，结果如图4所示。
从图4可以看出，XPS不仅证明了PDPC水凝胶中含有铜离子，即证明PDPC水凝胶铜离子的成功引入。此外，从XPS结果还可以看出，PDPC1水凝胶内含有两种价态的铜离子(Cu +和Cu 2+)，且定量分析两种铜离子的比例为Cu +：Cu 2+＝61.7:38.3，证明Cu +和Cu 2+之间的可逆的电子转移赋予其良好的抗氧化活性。
(4)利用应变振幅扫描测试实施例1中得到PDPC1水凝胶的弹性响应，测试了在0-1000％应变范围内PDPC1水凝胶的储能模量(G′)和损耗模量(G″)的改变，结果如图5所示。
从图5可以看出，在低应变条件下，G'和G”几乎保持不变，直到达到约100％，表明PDPC水凝胶可以维持较大的弹性变形。当应变进一步增加时，G'和G”急剧下降，在约400％时二者趋于相等，表明水凝胶网络结构破裂转变为溶胶状态。
(5)选择使用Anton Paar流变仪(MCR302,)测试实施例1中得到PDPC水凝胶黏度与剪切速率的关系以及可注射性，结果如图6所示。
从图6A可以看出，在0-100剪切速率范围内，PDPC的黏度随着剪切速率增加而减小，证明PDPC水凝胶具有剪切稀化性能，有明显的可注射性，且如图6B所示，水凝胶可以随注射器注射出来，也证明其具备良好的可注射性。
试验例2
测试实施例中制备得到PDPC水凝胶的体外粘接性能。
(1)测试实施例1中制备得到的PDPC水凝胶的组织粘接性能，结果如图7所示。
图7中分别表示PDPC水凝胶对于小鼠肾脏、肺和肝脏的粘接实例。从图7可以看出，PDPC水凝胶对小鼠各个部位均表现出较好的粘接性能。
(2)采用万能力学测试系统(MTS Criterion 43,MTS Criterion)测试实施例1中制备得到的PDP和PDPC水凝胶对猪皮和牛牙龈的粘接强度，结果如图8所示。从图中可以看出，PDPC水凝胶对猪皮肤和牛牙龈的粘接性能明显优于PDP水凝胶，证明PDPC水凝胶具备良好的粘接性能。
试验例3
(本试验例中所用对照组为不做任何处理的空白对照)
测试实施例中制备得到PDPC水凝胶的体外抗氧化性能。
(1)选用DPPH(MCE)作为ROS指示剂，将预先用无水乙醇配置好的DPPH加入EP管，再分组加入不同组份的水凝胶，黑暗中室温反应10min，测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶清除氧自由基的能力，结果如图9所示。图9中A表示清除氧自由基的原理图；B表示不同水凝胶与DPPH作用10min后的颜色变化图，C表示不同水凝胶与DPPH反应10min后清除氧自由基的定量结果。
利用DPPH的颜色变化进行分析，将DPPH与水凝胶反应10分钟记录颜色变化情况，并用酶标仪进行定量分析，如图9所示，PDPC水凝胶具备良好的清除氧自由基的能力，且PDPC2优于PDPC1水凝胶。
(2)选用NBT(Solarbio)作为超氧阴离子显色剂，该物质与超氧阴离子结合后会生成蓝色络合物。将提前配置好的黄嘌呤氧化酶、黄嘌呤和水凝胶分别加入双蒸水(ddwater)中，黑暗中反应以测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶清除超氧阴离子(O 2· -)的能力，结果如图10所示。图10中A表示清除超氧阴离子(O 2· -)的原理图；B表示不同水凝胶与NBT作用30min后的颜色变化图，C表示不同水凝胶与DPPH反应30min后清除O 2· -的定量结果。
图10可以看出，PDPC水凝胶具备良好的清除清除超氧阴离子的能力，相比于PDPC1水凝胶，PDPC2水凝胶的清除清除超氧阴离子的能力进一步提高。
(3)选用NBT(Sigma)作为羟基自由基的指示剂，该物质能被羟基自由基氧化生成蓝色产物。在NaAc/HAc缓冲液中加入提前配置好的FeSO 4溶液、H 2O 2和不同组份的水凝胶，常温避光反应5min，测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶清除羟基自由基(OH·)的能力，结果如图11所示。图11中A表示清除羟基自由基(OH·)的原理图；B表示不同水凝胶与TMB作用30min后的颜色变化图，C表示不同水凝胶与DPPH反应30min后清除OH·的定量结果。
图11可以看出，PDPC水凝胶具备良好的清除羟基自由基的能力，相较于PDPC1水凝胶，PDPC2水凝胶的清除羟基自由基的能力进一步提高。
(4)选用DCFH-DA作为胞内ROS指示剂，它本身无荧光，但能与ROS反应发出强烈的绿色荧光。将H 2O 2 与材料加入已经接种好L929细胞的孔板中，孵育24小时候加入DCFH-DA测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶对L929细胞的氧化应激改善能力，结果如图12所示。图12中A表示利用DCFH-DA探针检测不同水凝胶清除氧化应激状态L929细胞中ROS的荧光染色图；B表示对应的荧光强度。
图12可以看出，PDPC水凝胶具备更好的抵抗氧化应激的能力，相较于PDPC1水凝胶，PDPC2水凝胶的抵抗氧化应激的能力进一步提高。
(5)将不同组份的水凝胶与H 2O 2加入接种好L929细胞的孔板中孵育24h，然后加入用DMSO配置好的Ru(dpp) 3Cl 2，黑暗中孵育测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶促使L929细胞的氧气生成能力，结果如图13所示。图13中A表示利用Ru(dpp) 3Cl 2探针检测不同水凝胶与氧化应激状态L929细胞共培养后O 2生成情况的荧光染色图；B表示对应的荧光强度。
图13可以看出，可以看到相比于PDP而言，PDPC拥有更好的抗氧化性能，且与铜离子的含量成正比，即相较于PDPC1水凝胶，PDPC2水凝胶的抗氧化性能进一步提高。
试验例4
(本试验例中所用对照组为不做任何处理的空白对照)
测试实施例中制备得到PDPC水凝胶的体外生物相容性。
将不同组分不同浓度的水凝胶加入接种好L929细胞的孔板中共培养不同的时间点，测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶对L929细胞的毒性，结果如图14所示。图14中A表示不同浓度的不同水凝胶与L929细胞共培养1天后的细胞活性；B表示不同浓度的不同水凝胶与L929细胞共培养3天后的细胞活性；C表示不同浓度的不同水凝胶与L929细胞共培养7天后的细胞活性。
图14可以看出，不同浓度的不同水凝胶与L929细胞共培养1天、3天和7天后的细胞活性较空白对照组而言均无明显差异，证明不同水凝胶均具备良好的生物相容性。
试验例5
(本试验例中所用对照组为不做任何处理的空白对照)
测试实施例中制备得到PDPC水凝胶的光热性能。
(1)采用808nm近红外激光发射器，以相同的高度相同的功率(1W/m 2)及相同的时间(5min)照射不同组份的水凝胶，测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶光热性能，结果如图15所示。图15中A表示不同水凝胶在近红外光照射6min内的温度热图；B表示不同水凝胶在近红外光照射6min内的温度变化定量结果。
图15可以看出，不同PDPC水凝胶均拥有优异的光热性能，PDPC2水凝胶的温度从最开始的22℃升至51℃，且快速趋于稳定到达平台期。
(2)测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶光热稳定性，结果如图16所示。
图16表示PDPC水凝胶在相同条件进行4个循环激光照射周期的升温测试，在4个不同周期内PDPC水凝胶均呈现良好的光热性能，证实了PDPC水凝胶具备优异的光稳定性。
试验例6
(本试验例中所用对照组为不做任何处理的空白对照)
测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶抗菌性能，结果如图17-图20所示。
(1)图17为不同水凝胶分别与大肠杆菌(E.C)(A)和金黄色葡萄球菌(S.A)(B)共培养24h后的菌落图，证明PDPC水凝胶可以抑制两种菌的菌落形成；
(2)图18为不同水凝胶分别与E.C(A)和S.A(B)共培养24h后的细菌活死染色(Live/Dead双染色试剂盒Sigma)图及其定量结果，证明PDPC水凝胶可以有效杀死两种细菌；
(3)图19为不同水凝胶分别与E.C(A)和S.A(B)共培养24h后的结晶紫染色及定量结果图，证明PDPC水凝胶可以有效杀灭细菌生物膜；
(4)图20为不同水凝胶分别与E.C(A)和S.A(B)细菌生物膜共培养48h后的荧光染色图，也证明PDPC水凝胶可以有效杀灭细菌生物膜。
从以上结果均可以看出，PDPC水凝胶均具备更加优异的抗菌效果，且PDPC2水凝胶优于PDPC1水凝胶。
试验例7
(本试验例中所用对照组为不做任何处理的空白对照)
测试实施例1和实施例2制备得到的PDP、PDPC1和PDPC2水凝胶的光热抗菌性能，结果如图21-图24所示。
(1)图21为不同水凝胶分别与大肠杆菌(E.C)(A)和金黄色葡萄球菌(S.A)(B)共培养24h并用808nm近红外光照射5min后的菌落图，证明PDPC水凝胶光照后可进一步抑制两种菌的菌落形成。
(2)图22为不同水凝胶分别与E.C(A)和S.A(B)共培养24h并用808nm近红外光照射5min后的细菌活死染色图及其定量结果，证明PDPC水凝胶光照后可更加有效杀死两种细菌。
(3)图23为不同水凝胶分别与E.C(A)和S.A(B)共培养24h并用808nm近红外光照射5min后的结晶紫染色及定量结果图，证明PDPC水凝胶光照后可以有效杀灭细菌生物膜；
(4)图24为不同水凝胶分别与E.C(A)和S.A(B)细菌生物膜共培养48h并用808nm近红外光照射5min后的荧光染色图，也证明PDPC水凝胶光照后可以有效杀灭细菌生物膜。
从以上结果均可以看出，PDPC水凝胶均具备更加优异的光热抗菌效果，且PDPC2水凝胶优于PDPC1水凝胶。
试验例8
(本试验例中所用对照组为沪士达商用明胶海绵。)
测试实施例2制备得到的PDPC2水凝胶的止血性能，结果如图25-图31所示。
(1)图25为PDPC水凝胶对SD大鼠肝脏止血定量结果，图25中A表示60s内肝脏的出血情况图，B表示60s内肝脏的失血量定量结果。如图所示，PDPC水凝胶具备更优异的止血性能，可使大鼠肝脏快速止血，且相较于空白组的失血量明显减少；
(2)图26为PDPC水凝胶对SD大鼠心脏止血过程图，从图中可以看出，使用PDPC2水凝胶对大鼠心脏创口进行涂抹后PDPC2水凝胶可快速有效封闭心脏创口，使心脏停止出血，证明其优异的止血性能；
(3)图27为PDPC水凝胶对新西兰大白兔颈动脉止血过程图，如图所示，将颈动脉截流并制造创口，然后用PDPC2水凝胶涂抹创口，松开动脉夹后PDPC2水凝胶可成功封闭猪颈动脉的创口出血，且在原创口的远心端再建立一个创口后，颈动脉可恢复血供，表明材料应用后未影响兔子的正常血供。
(4)图28为PDPC水凝胶对巴马小型猪的肝脏线性出血伤口的止血过程图；图29为PDPC水凝胶对巴马小型猪的肝脏圆形出血伤口的止血过程图。图28和图29均可以看出，使用PDPC2水凝胶覆盖猪肝脏线性和圆形出血创口均可快速成功封闭创口，并达到止血的目的。证明PDPC水凝胶可封闭猪等大型动物的出血，具备优异的止血性能。
(5)图30为PDPC水凝胶对巴马小型猪心脏止血过程图；图31为PDPC水凝胶对巴马小型猪颈动脉止血过程图。同上，PDPC水凝胶不仅可以快速封闭大鼠、兔子等中小型动物心脏和颈动脉的出血，还可以封闭猪等大型动物心脏和颈动脉的出血，即使在心脏和颈动脉较大的血液压力冲击下仍旧能够牢固黏附于创口组织表面，达到快速的止血效果。
试验例9
(本试验例中所用对照组为不做任何处理的空白对照)
测试实施例2的PVA、制备得到的PDP以及PDPC2水凝胶对小鼠皮肤伤口的愈合效果，结果如图32和图33所示。
图32为PDPC水凝胶对BALBc小鼠背部皮肤伤口愈合过程图，并通过H&E和天狼星红染色验证水凝胶促进创口胶原纤维形成，进一步选用荧光染色探针标记α-SMA、CD31、iNOS和CD206，经由Nikon,Ci-POL荧光显微镜观测来验证PDPC水凝胶减轻创口炎症，促进创口血管生成，如图所示，PDPC水凝胶较其他组别的小鼠伤口愈合更快，证明PDPC水凝胶可有效促进皮肤伤口愈合。图33为不同组别水凝胶处理小鼠背部皮肤伤口后在伤口处提取菌液的菌落图，如图所示，PDPC水凝胶处理小鼠皮肤伤口后可明显减少伤口处的细菌数量，证明PDPC水凝胶具备良好的抗菌性能，从而促进皮肤伤口愈合。
试验例10
(本试验例中所用对照组为不做任何处理的空白对照)
测试实施例1和实施例2制备得到的PDP和PDPC2水凝胶的体内生物相容性，结果如图34所示。图34分别表示不同组别水凝胶处理后的白细胞(WBC)(A)、红细胞(RBC)(B)、血小板(PLT)(C)、血清肌酸激酶(CK)(D)、乳酸脱氢酶(LDH)(E)、谷丙转氨酶(ALT)(F)、谷草转氨酶(AST)(G)、尿素氮(BUN)(H)和肌酐 (CREA)(I)。
如图34中A-C所示，不同组别水凝胶处理伤口10天后的WBC、RBC和PLT与空白对照组均无明显差异，证明PDPC水凝胶无明显的血液毒性；如图34中D-I所示，不同组别水凝胶处理伤口10天后的CK、LDH、ALT、AST、BUN和CREA与空白对照组均无明显差异，证明PDPC水凝胶对心脏、肝脏、肾脏均无明显毒性。证明PDPC水凝胶具备优异的体内生物相容性。
试验例11
测试实施例1的PDP、实施例1和实施例2分别制备得到的PDPC1和PDPC2水凝胶和对比例1制备得到的PDPC3水凝胶的溶血性，结果如图35所示。
从图35可以看出，PDP、PDPC1和PDPC2水凝胶组颜色与阴性对照组(PBS)相似，表明其均未发生明显溶血，而PDPC3水凝胶组与阳性对照组(TritonX-100)相似，呈现亮红色，表明其发生严重溶血，以上结果证明PDP、PDPC1和PDPC2水凝胶均具备良好的生物相容性，而PDPC3水凝胶生物相容性较差，无法进一步应用于生物医学领域。
试验例12
测试实施例2制备得到PDPC2水凝胶与对比例2中常见市售的明胶海绵(merocel组)对伤口止血的效果，结果如图25所示。
从图25可以看出，PDPC2水凝胶的止血效果明显优于明胶海绵的止血效果，其出血量明显少于明胶海绵，且差异具备统计学意义，证明PDPC水凝胶比市售明胶海绵可以达到更好的止血效果，减少病人出血量，加速病人伤口愈合。
试验例13
测试实施例1中使用CuCl 2·2H 2O与对比例3中CuSO 4制备PDPC水凝胶的效果，结果如图36所示。
从图36中可以看出，将PDP溶液与CuCl 2·2H 2O溶液混合后的PDPC为均匀分布的溶液状态，而将PDP溶液与CuSO 4溶液混合后的PDPC出现明显的沉淀，即CuSO 4溶液无法与PDP溶液充分反应形成均匀的PDPC溶液，也无法形成所需的PDPC水凝胶，因此本公开选取CuCl 2·2H 2O制备PDPC水凝胶。
综上所述，本公开提供一种伤口敷料的制备方法、伤口敷料及应用，以聚乙烯醇(PVA)为载体，整合多巴(DOPA)优良的湿粘接性能和金属离子Cu的优良特性，实现儿茶酚基的动态交联，制备了一种仿生的贻贝多功能水凝胶(PDPC水凝胶)。该水凝胶具备以下优良特性：
(1)超强的粘接性能，在血液的冲洗及病人运动情况下能够长期稳固粘附于创伤组织；
(2)超强、快速的止血性能，即使在心脏、颈动脉等血管压力下仍旧能够快速有效止血，降低死亡率；
(3)良好的可注射性和塑形能力，操作便捷，适用于不同形状缺损；
(4)高效、广谱的抗氧化活性；
(5)优异的近红外光热响应性和抗菌效果；
(6)良好的生物安全性能和血液相容性；
(7)有效促进伤口愈合。
以上仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。
工业实用性
本公开提供了一种伤口敷料，具有丰富的多孔结构，可通过快速吸收血液对伤口进行止血，是一种集组织粘附性、快速止血特性、ROS清除活性、近红外光热响应性、抗菌性等多功能于一体的材料，能够牢固粘附于伤口表面快速止血、保护创面并发挥抗炎效应，发挥高效的抗菌效果，快速促进创面伤口的愈合，且本公开制备方法简便，易规模化生产，因此本公开的伤口敷料及其制备方法均具备广泛的临床应用前景。

Claims (14)

  1. 一种伤口敷料的制备方法，其特征在于，包括：利用聚乙烯醇与3,4-二羟基苯丙氨酸反应得到PDP聚合物，将所述PDP聚合物与铜离子络合形成水凝胶。
  2. 根据权利要求1所述的制备方法，其特征在于，包括：
    将聚乙烯醇溶解之后，在催化剂存在的条件下与3,4-二羟基苯丙氨酸反应，在反应完成之后通过后处理得到凝胶状态的PDP聚合物；
    将所述PDP聚合物复溶之后与铜盐混合反应，以使所述PDP聚合物与铜离子络合。
  3. 根据权利要求2所述的制备方法，其特征在于，所述聚乙烯醇与所述3,4-二羟基苯丙氨酸的摩尔比为2.5-4.5:1；优选为3-4:1；
    优选地，所述聚乙烯醇的分子量为85-124kDa。
  4. 根据权利要求2或3所述的制备方法，其特征在于，所述催化剂为NaHSO 4·H 2O；
    优选地，将所述聚乙烯醇在90-110℃的条件下溶解于有机溶剂中，所述有机溶剂为二甲基亚砜。
  5. 根据权利要求2-4中任一项所述的制备方法，其特征在于，所述聚乙烯醇和所述3,4-二羟基苯丙氨酸的反应时间为20-24h，反应温度为75-80℃；
    优选地，反应过程在保护气存在的条件下进行。
  6. 根据权利要求2-5中任一项所述的制备方法，其特征在于，所述后处理的过程包括：将反应后的溶液进行透析、旋蒸和干燥；
    优选地，透析时间为2-4h；
    优选地，所述干燥采用冻干的方式，冻干时间为1-2天。
  7. 根据权利要求2-6中任一项所述的制备方法，其特征在于，将所述PDP聚合物在加热的条件下与水混合溶解得到PDP溶液，在所述PDP溶液中滴加铜盐溶液，反应30-60min；
    优选地，控制所述PDP溶液中PDP聚合物浓度为80-120mg/mL。
  8. 根据权利要求7所述的制备方法，其特征在于，所述铜盐选自氯化铜、硫酸铜和硝酸铜中的至少一种；
    优选地，所述铜盐为CuCl 2·2H 2O，且所述PDP聚合物与所述铜盐的质量比为100:1-2。
  9. 一种伤口敷料，其特征在于，通过权利要求1-8中任一项所述的制备方法制备而得。
  10. 权利要求9所述的伤口敷料在制备组织创伤修复材料中的应用。
  11. 根据权利要求9所述的伤口敷料，用于修复组织创伤的用途。
  12. 一种对受试者中创伤处理的方法，包括：
    向所述有此需要的受试者施用根据权利要求9所述的伤口敷料。
  13. 根据权利要求12的方法，其中，所述受试者患有体表创伤、静脉损伤、动脉损伤或经历了手术。
  14. 根据权利要求13的方法，其中，所述手术是与心脏、肝脏、脾脏或肾脏相关的手术。
PCT/CN2023/070596 2022-04-13 2023-01-05 一种伤口敷料的制备方法、伤口敷料及应用 WO2023197700A1 (zh)

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CN115975224B (zh) * 2023-03-16 2023-08-08 四川大学 一种pH/ROS双响应的组织粘附载药水凝胶及其制备方法和应用

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