Polyphenol-active protein coating for chronic wound treatment and preparation method thereof

Abstract

The invention belongs to the technical field of biological materials, and particularly relates to a polyphenol-active protein coating for chronic wound treatment and a preparation method of the polyphenol-active protein coating. The preparation method comprises the following steps: firstly, forming carboxylated negative charge surfaces on different dressing surfaces by utilizing non-material dependence of a polydopamine coating; and then fixing polyphenol-protein composite nanoparticles which are rich in multiple active sites and carry positive charges on the surface of the dressing by utilizing electrostatic interaction. According to the preparation method, coatings can be directly constructed on the surfaces of different substrate dressing materials such as gauze, cotton pads, transparent dressings, electrostatic spinning membranes, chitosan dressings and polyurethane foam, and different polyphenols and proteins are selectively adsorbed on the surfaces of the dressings according to different stages of wound healing; the surface has a specific or broad-spectrum biological function, and the biological safety and the biological functionality of the chronic wound dressing are better met.

Description

Technical Field

[0001] The present invention belongs to the field of biomaterials, and in particular relates to a polyphenol-active protein coating for treating chronic wounds and a preparation method thereof. Background Art

[0002] Diabetes is a global health problem, with its prevalence and incidence continuing to rise. According to the International Diabetes Federation (IDF), there were approximately 537 million adults with diabetes worldwide in 2021. Diabetes can cause a variety of serious complications, including cardiovascular disease, neuropathy, nephropathy, and chronic wounds. Approximately 15% to 25% of diabetic patients will develop chronic wounds, and 66% of patients will experience ulcer recurrence within 5 years after healing. Unlike acute wounds, which heal autonomously through the four stages of hemostasis, inflammation, proliferation, and remodeling, the healing process of diabetic wounds is more complex and difficult due to multiple factors such as a high-sugar microenvironment, chronic inflammation, excessive oxidative stress, impaired cellular function, and a high risk of infection. These factors are inherently interconnected, intertwined, and exacerbated, putting patients at risk of amputation or even death. Therefore, research on the treatment and healing mechanisms of diabetic wounds is a major clinical challenge that urgently needs to be addressed.

[0003] As one of the effective means for diabetic wound management, wound dressings can improve the treatment effect by protecting the wound, reducing infection, maintaining a moist environment, etc. Currently, gauze, cotton pads, transparent dressings, silver ion dressings, silicone dressings, etc. are widely used in clinical practice, but there are still problems such as easy adhesion to the wound causing secondary injury, poor breathability, non-absorbency, possible induction of drug resistance or cytotoxicity during long-term use, and high cost. Moreover, a single basic dressing also faces multiple challenges in regulating the complex microenvironment of diabetic wounds. Therefore, functional modification of basic dressings through surface modification technology to precisely meet the multi-dimensional needs of diabetic wounds has important research value. The biomimetic strategy in nature provides important inspiration for material design - research shows that polyphenol-protein complexes naturally present in plants usually have antibacterial, antioxidant, anti-inflammatory, and structure-strengthening properties, which endow plants with the ability to resist biological threats and promote wound healing. Surface engineering strategies have been widely studied as an effective means to endow the surface of biomedical materials / devices with multi-functional activities. Therefore, preparing a polyphenol-protein coating on the surface of the wound dressing substrate material through surface modification technology is of great significance for promoting wound healing. However, the current methods for preparing polyphenol-protein surfaces mainly include: 1) Layer-by-layer self-assembly method - by alternately depositing polyphenols and protein molecules with opposite charges and using electrostatic attraction to construct the coating layer by layer; 2) Covalent cross-linking method - forming a stable coating through chemical cross-linking agents (such as genipin, glutaraldehyde) or oxidative coupling reactions (after polyphenols are oxidized to quinones and react with proteins); 3) Physical adsorption after blending - directly coating a film on the material surface after mixing polyphenol and protein solutions; 4) Electrochemical deposition method; 5) Microfluidic / 3D printing technology. These methods can form polyphenol-protein coatings on the surfaces of different substrate materials according to requirements. However, they also face some common problems and challenges, such as insufficient stability, time-consuming processes, loss of substance activity, potential toxicity, high environmental sensitivity, low loading capacity, high equipment cost, low technology maturity, etc., which greatly limit the large-scale or commercial application of polyphenol-protein coatings in the field of chronic wound dressings.

[0004] Therefore, based on the above problems existing at the present stage, the technical solution of the present invention is proposed. Summary of the Invention

[0005] In order to solve the problems existing in the prior art, the present invention provides a polyphenol-active protein coating for the treatment of chronic wounds and a preparation method thereof. The preparation method can directly construct a polyphenol-protein coating on the surfaces of different wound dressing substrate materials (such as gauze, cotton pads, transparent dressings, electrospun membranes, chitosan dressings, polyurethane foams), and selectively adsorb different polyphenols and proteins on the surface according to the healing situation of the wound, so that the surface has specific or broad-spectrum biological functions, better meeting the biosafety and biological functionality of wound dressings.

[0006] The solution of the present invention is to provide a preparation method of a polyphenol-active protein coating for chronic wound treatment, and the preparation method includes the following steps: (1) Dissolve dopamine, carboxyl compound and oxidant in acidic buffer solution respectively to obtain dopamine solution, carboxyl compound solution and oxidant solution; (2) Mix the dopamine solution, carboxyl compound solution and oxidant solution to obtain a mixed solution; then completely immerse the wound dressing base material in the mixed solution for reaction and wash thoroughly to obtain a base material with negatively charged surface carboxylation; (3) Dissolve water-insoluble polyphenol and protein in PBS solution containing DMSO respectively to obtain polyphenol solution and protein solution. Under the magnetic stirring environment, drop the polyphenol solution into the protein solution and continuously stir for reaction to obtain a suspension of polyphenol-protein composite nanoparticles; (4) Immerse the base material with negatively charged surface carboxylation in the suspension of polyphenol-protein composite nanoparticles for reaction and wash thoroughly to obtain the polyphenol-active protein coating for chronic wound treatment.

[0007] For the convenience of understanding the present invention, the reaction principle of the present invention is explained: In the preparation method of the present invention, first, dopamine and a carboxyl compound undergo oxidation, cross-linking, and polymerization reactions under the action of an oxidant to form a negatively charged carboxylated surface on the surface of wound dressing substrates with different shapes and materials. Subsequently, a PBS solution containing DMSO is used to dissolve the water-insoluble polyphenol compound and protein respectively. The polyphenol compound and protein achieve intermolecular multi-scale bonding under the single action or dynamic cooperation of hydrogen bonding, hydrophobic interaction, π-π stacking effect, electrostatic attraction, and van der Waals force, and finally self-assemble to form polyphenol-protein composite nanoparticles with a stable structure. The polyphenol-protein composite nanoparticles are rich in multiple active sites and carry positive charges. When they come into contact with the negatively charged surface modified with carboxyl groups, the polyphenol-protein composite nanoparticles are in-situ anchored through an electrostatic-driven self-assembly strategy, thereby forming a stable superhydrophilic polyphenol-active protein coating on the surface of the chronic wound dressing. It should be noted that the formation and fixation processes of the polyphenol-protein composite nanoparticles are mainly achieved through non-covalent bonds, and their mild bonding strategy completely preserves the phenolic hydroxyl active sites of the polyphenol and the native conformation of the protein. Based on the precise cooperation of functional modules (the triple effect of antioxidant-antibacterial-immune regulation of polyphenols and the dual cooperation of antibacterial-wound healing promotion of proteins), the multi-level integration of active ingredients is systematically realized, and finally a chronic wound repair dressing system with a polyphenol-protein multi-level biological cooperation mechanism is constructed. In addition, since the binding and fixation of polyphenol-protein are mainly achieved through electrostatic attraction, hydrogen bond network, hydrophobic interaction, etc. on the coating surface, the polyphenol-active protein coating on the dressing surface has pH responsiveness and will accelerate the release of polyphenols and active proteins in the weakly acidic (pH≈5.5~6.5) microenvironment formed by bacterial infection, and timely adjust the stability of the microenvironment.

[0008] Preferably, in step (1), the acidic buffer solution is one of acetic acid-sodium acetate buffer solution, 2-(N-morpholino)ethanesulfonic acid buffer solution, glycine-hydrochloric acid buffer solution, phthalic acid-hydrochloric acid buffer solution, potassium hydrogen phthalate-sodium hydroxide buffer solution, disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, and citric acid-sodium citrate buffer solution.

[0009] Preferably, in step (1), the carboxyl compound is one or a combination of two or more of lactic acid, malic acid, tartaric acid, glycine, glutamic acid, α-ketoglutaric acid, vitamin C, acetylsalicylic acid, oxalic acid, phthalic acid, and salicylic acid.

[0010] Preferably, in step (1), the oxidant is one or a combination of two or more of hydrogen peroxide, ammonium persulfate, concentrated nitric acid, sodium periodate, potassium permanganate, and potassium dichromate.

[0011] Preferably, in step (2), the wound dressing base material is one of gauze, cotton pad, transparent dressing, electrospun membrane, chitosan dressing, and polyurethane foam.

[0012] Preferably, in step (3), the water-insoluble polyphenol is one or a combination of two or more of catechin, catechin-3-gallucoside, catechin-4'-gallucoside, limocitrin, quercetin flavanone, hesperetin flavanone, soy isoflavone, flavanone, liquiritigenin, oleuropein, hydroxytyrosol, caffeophenol ester, procyanidin, quercetin, anthocyanin, magnolol, and honokiol.

[0013] Preferably, in step (3), the protein is one or a combination of two or more of growth factor proteins, structural proteins and extracellular matrix-related proteins, antimicrobial peptides and immunomodulatory peptides, synthetic peptides and biomimetic peptides; and / or, the growth factor proteins are one or a combination of two or more of epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, and transforming growth factor-β; and / or, the structural proteins and extracellular matrix-related proteins are one or a combination of two or more of collagen, fibronectin, laminin, albumin, fibrinogen, type I collagen, type II collagen, type III collagen, type IV collagen, and lysozyme; and / or, the antimicrobial peptides and immunomodulatory peptides are one or a combination of two or more of LL-37, α-defensin, β-defensin, Tβ4, lactoferrin, QQKFQFQFEQQ peptide, and KLAKLAK peptide; and / or, the synthetic peptides and biomimetic peptides are one or a combination of two or more of RGD peptide, copper peptide GHK-Cu, and lipopeptide PXL01.

[0014] Preferably, in step (1), the pH value of the acidic buffer solution is 2 to 6.5; and / or, in step (1), the mass concentrations of the dopamine solution, carboxyl compound solution, and oxidant solution are 0.5 to 50 mg / mL, 0.5 to 20 mg / mL, and 0.5 to 20 mg / mL, respectively; and / or, in step (3), the volume concentration of DMSO in the PBS solution containing DMSO is 0.5 to 10%; and / or, in step (3), the mass concentrations of the water-insoluble polyphenol solution and the protein solution are 0.1 to 10 mg / mL and 1 to 100 mg / mL, respectively; Preferably, in step (2), the reaction time is 0.5 to 24 h, and the reaction temperature is 15 to 40 °C; And / or, in step (3), the reaction time is 1 to 48 h, the stirring speed is 600 to 800 rpm, and the reaction temperature is 10 to 37°C; And / or, in step (4), the reaction time is 0.5 to 48 h, and the reaction temperature is 10 to 37°C.

[0015] Based on the same inventive concept, another object of the present invention is to provide a polyphenol-active protein coating for the treatment of chronic wounds obtained by the above preparation method.

[0016] The beneficial effects of the present invention are as follows: 1. The active protein coating has excellent biocompatibility and biological functionality and is widely used in the field of surface modification of biomaterials. At present, proteins are mainly modified onto the material surface by physical adsorption, chemical cross-linking, and layer-by-layer self-assembly. However, there is still a problem that it is difficult to achieve both protein activity and stability. In the preparation method of the present invention, a carboxylated negatively charged surface is first prepared, which has excellent hydrophilicity and micro-nano topological structure; subsequently, positively charged polyphenol-protein composite nanoparticles are stably fixed onto the dressing surface through electrostatic adsorption. During the whole process, the binding and fixation of proteins are mainly non-covalent bonds, effectively preserving the structure and function of proteins.

[0017] 2. Polyphenols are natural active small molecules with excellent biological functions such as antioxidant, antibacterial, and inflammation regulation. However, the phenolic hydroxyl groups of polyphenols are easily oxidized, resulting in a decrease in their antioxidant capacity, inflammation regulation, and other biological functions. In the preparation method of the present invention, polyphenols and proteins are combined in a non-covalent manner, effectively slowing down the oxidation of phenolic hydroxyl groups and retaining the biological functions of polyphenols.

[0018] 3. The microenvironment of chronic wounds is difficult to heal due to factors such as excessive oxidative stress, inflammatory disorders, metabolic abnormalities, microbial infections, and cell dysfunction; a single functional protein or active molecule cannot effectively cope with the adverse factors in the microenvironment. The preparation method of the present invention is based on the precise coordination of functional modules (the three-in-one effect of antioxidant-antibacterial-immune regulation of polyphenols and the dual coordination of antibacterial-promoting healing of proteins), and systematically realizes the multi-level integration of the active ingredient polyphenols and proteins.

[0019] 4. Chronic wounds are prone to bacterial infections and the microenvironment is weakly acidic (pH ≈ 5.5 - 6.5). Through various mechanisms such as inhibiting the proliferation and migration of repair cells, inhibiting enzyme activity, and aggravating the inflammatory response, the tissue repair process is hindered. Therefore, timely prevention of bacterial infection is crucial for the healing of chronic wounds. The binding and fixation of polyphenols - proteins in the preparation method described in the present invention are mainly achieved through coating electrostatic attraction, hydrogen bond network, hydrophobic interaction, etc. Therefore, the polyphenol - active protein coating on the dressing surface has pH responsiveness and will accelerate the release of polyphenols and active proteins in the weakly acidic microenvironment formed by bacterial infection, timely sterilize, and regulate the stability of the microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0021] Figure 1 It is the Zeta potential diagram of the carboxylated surface and the polyphenol - protein composite nanoparticle suspension in the polyphenol - active protein coating prepared for the treatment of chronic wounds in Example 1.

[0022] Figure 2 It is the scanning electron microscope image of the polyphenol - active protein coating prepared for the treatment of chronic wounds in Example 1.

[0023] Figure 3 It is the elemental content result diagram of the XPS detection of the polyphenol - active protein coating prepared for the treatment of chronic wounds in Example 2.

[0024] Figure 4 It is the full - spectrum diagram of the XPS detection of the polyphenol - active protein coating prepared for the treatment of chronic wounds in Example 2.

[0025] Figure 5 It is the laser confocal image of the surface macrophage cytoskeleton staining of the polyphenol - active protein coating prepared for the treatment of chronic wounds in Example 3.

[0026] Figure 6 It is the antibacterial rate of the polyphenol - active protein coating prepared for the treatment of chronic wounds in Example 4. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0027] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without any creative work fall within the scope protected by the present invention.

[0028] Example 1

[0029] This example provides a preparation method for a polyphenol-active protein coating for wound treatment. The preparation method is as follows: (1) Prepare an acetic acid-sodium acetate buffer solution with a pH of 3, dissolve dopamine, malic acid and potassium permanganate oxidant to form a dopamine solution, a malic acid solution and a potassium permanganate solution with mass concentrations of 6 mg / mL, 6 mg / mL and 6 mg / mL respectively; (2) Mix the dopamine solution, malic acid solution and potassium permanganate solution prepared in (1) in equal volumes, completely immerse the transparent film wound dressing in the mixed solution, and carry out a constant temperature reaction at 25 °C for 2 hours, and wash thoroughly to obtain a transparent film wound dressing with negatively charged carboxylated surface; (3) Dissolve magnolol and lysozyme in a PBS solution containing 1% DMSO by volume concentration to obtain a magnolol solution and a lysozyme solution with mass concentrations of 4 mg / mL and 40 mg / mL respectively. Under a magnetic stirring environment, drop an equal volume of the magnolol solution into the lysozyme solution, and continuously stir the mixed solution at a speed of 600 rpm for 5 hours until a stable and uniform magnolol-lysozyme composite nanoparticle suspension is formed, and set aside; (4) Immerse the transparent film wound dressing with negatively charged carboxylated surface prepared in (2) in the magnolol-lysozyme composite nanoparticle suspension prepared in (3), carry out a constant temperature reaction at 25 °C for 10 hours, and wash thoroughly to obtain a magnolol-lysozyme coating for chronic wound treatment.

[0030] Example 2

[0031] This example provides a preparation method for a polyphenol-active protein coating for chronic wound treatment. The preparation method is as follows: (1) Prepare a glycine-hydrochloric acid buffer solution with a pH of 5, dissolve dopamine, glycine and sodium periodate oxidant to form a dopamine solution, a glycine solution and a sodium periodate solution with mass concentrations of 9 mg / mL, 6 mg / mL and 9 mg / mL respectively; (2) Mix the dopamine solution, glycine solution, and sodium periodate solution prepared in (1) in equal volumes. Immerse the electrospun membrane wound dressing completely in the mixture, and react at a constant temperature of 20 °C for 5 hours. Then wash it thoroughly to obtain an electrospun membrane wound dressing with negatively charged carboxylated surface. (3) Dissolve quercetin and type IV collagen in PBS solution containing 5% DMSO by volume to obtain quercetin solution and type IV collagen solution with mass concentrations of 1 mg / mL and 10 mg / mL respectively. Under a magnetic stirring environment, drop an equal volume of quercetin solution into the type IV collagen solution, and continuously stir the mixture at a speed of 650 rpm for 8 hours until a stable and uniform quercetin-type IV collagen composite nanoparticle suspension is formed. Set it aside for later use. (4) Immerse the electrospun membrane wound dressing with negatively charged carboxylated surface prepared in (2) in the quercetin-type IV collagen composite nanoparticle suspension prepared in (3), and react at a constant temperature of 20 °C for 20 hours. Then wash it thoroughly to obtain a quercetin-type IV collagen coating for the treatment of chronic wounds.

[0032] Example 3

[0033] This example provides a preparation method for a polyphenol-active protein coating for the treatment of chronic wounds. The preparation method is as follows: (1) Prepare a disodium hydrogen phosphate-citric acid buffer solution with a pH of 5.5, and dissolve dopamine, salicylic acid, and ammonium persulfate oxidant to form a dopamine solution, a salicylic acid solution, and an ammonium persulfate solution with mass concentrations of 3 mg / mL, 6 mg / mL, and 6 mg / mL respectively. (2) Mix the dopamine solution, salicylic acid solution, and ammonium persulfate solution prepared in (1) in equal volumes. Immerse the polyurethane foam wound dressing completely in the mixture, and react at a constant temperature of 30 °C for 2 hours. Then wash it thoroughly to obtain a polyurethane foam wound dressing with negatively charged carboxylated surface. (3) Dissolve procyanidin and antibacterial peptide QQKFQFQFEQQ in PBS solution containing 1.5% DMSO by volume to obtain procyanidin solution and antibacterial peptide QQKFQFQFEQQ solution with mass concentrations of 4 mg / mL and 20 mg / mL respectively. Under a magnetic stirring environment, drop an equal volume of procyanidin solution into the antibacterial peptide QQKFQFQFEQQ solution, and continuously stir the mixture at a speed of 700 rpm for 12 hours until a stable and uniform procyanidin-antibacterial peptide composite nanoparticle suspension is formed. Set it aside for later use. (4) Immerse the surface carboxylated negatively charged polyurethane foam wound dressing prepared in (2) in the procyanidin - antimicrobial peptide composite nanoparticle suspension prepared in (3), react at a constant temperature of 30 °C for 2 hours, and wash thoroughly to obtain a procyanidin - antimicrobial peptide coating for the treatment of chronic wounds.

[0034] Example 4

[0035] This example provides a method for preparing a polyphenol - active protein coating for the treatment of chronic wounds. The preparation method is as follows: (1) Prepare a citric acid - sodium hydroxide - hydrochloric acid buffer solution with a pH of 4, dissolve dopamine, α - ketoglutaric acid, and potassium dichromate oxidant to form a dopamine solution, an α - ketoglutaric acid solution, and a potassium dichromate solution with mass concentrations of 12 mg / mL, 12 mg / mL, and 6 mg / mL respectively; (2) Mix the dopamine solution, α - ketoglutaric acid solution, and potassium dichromate solution prepared in (1) in equal volumes, completely immerse the gauze wound dressing in the mixture, react at a constant temperature of 20 °C for 12 hours, and wash thoroughly to obtain a transparent film wound dressing with a surface carboxylated negative charge; (3) Dissolve honokiol and fibroblast growth factor in a PBS solution containing 1% DMSO by volume concentration to obtain a honokiol solution and a fibroblast growth factor solution with mass concentrations of 6 mg / mL and 100 ng / mL respectively. Under a magnetic stirring environment, drop an equal volume of the honokiol solution into the fibroblast growth factor solution, and continuously stir the mixture at a speed of 600 rpm for 1 hour until a stable and uniform honokiol - fibroblast growth factor composite nanoparticle suspension is formed for standby; (4) Immerse the transparent film wound dressing with a surface carboxylated negative charge prepared in (2) in the honokiol - fibroblast growth factor composite nanoparticle suspension prepared in (3), react at a constant temperature of 20 °C for 15 hours, and wash thoroughly to obtain a honokiol - fibroblast growth factor coating for the treatment of chronic wounds.

[0036] Verification Example Figure 1 To measure the Zeta potential of the carboxylated surface and the polyphenol - protein composite nanoparticle suspension in the polyphenol - active protein coating prepared for the treatment of chronic wounds in Example 1, it can be seen from Figure 1 that the Zeta potentials of the prepared carboxylated surface and the polyphenol - protein composite nanoparticle suspension are approximately - 43 mV and 28 mV respectively, indicating that the carboxylated surface mainly fixes the polyphenol - protein composite nanoparticles on the dressing surface through electrostatic interaction, effectively retaining the biological activities of polyphenols and proteins.

[0037] Experimental method: Trim the transparent film dressing into a rectangle of 1×2 cm (the size requirement of the solid surface Zeta potential tester for the test sample), and set aside. According to steps (1) and (2) in the preparation method described in Example 1, deposit a carboxylated surface on the surface of the 1×2 cm rectangular transparent dressing. Subsequently, prepare a polyphenol-protein composite nanoparticle suspension according to step (3) in the preparation method described in Example 1. Finally, use a solid surface Zeta potential tester (model: SurPASS) and a nanoparticle size and Zeta potential analyzer (model: Zeta sizer Nano ZS) to detect the charge values of the carboxylated surface and the polyphenol-protein composite nanoparticle suspension.

[0038] Figure 2 Figure 4 shows the SEM surface morphology of the polyphenol-active protein coating prepared in Example 1 for the treatment of chronic wounds. As Figure 2 shown, the carboxylated substrate surface presents a micro-nano composite topological morphology. After being modified with polyphenol-protein composite nanoparticles, the substrate surface is uniformly coated, confirming that the polyphenol-protein composite nanoparticles are successfully grafted onto the carboxylated surface, realizing the controllable construction of the active protein coating.

[0039] Experimental method: Trim the transparent dressing into a square of 0.8×0.8 cm (this size is used for field emission scanning electron microscopy detection), and mark the front and back of the sample at the same time, and set aside. Prepare carboxylated transparent dressings and polyphenol-active protein coating transparent dressings respectively according to the preparation method described in Example 1. Naturally dry the prepared two surfaces, spray gold (spray gold time: 90 seconds), and then observe the SEM morphology of different sample surfaces with a field emission scanning electron microscope (model: S-4800).

[0040] Figure 3 Figure 5 shows the elemental content results of the XPS detection of the polyphenol-active protein coating prepared in Example 2 for the treatment of chronic wounds. It can be seen from Figure 3 the figure that compared with the chemical elements of the carboxylated surface, the polyphenol-active protein coating has an S element with a content of 0.94%, and the N element in the coating increases significantly, while the C element content decreases. The S element is a unique element of protein different from polyphenol, further indicating that the protein is successfully grafted onto the carboxylated surface.

[0041] Experimental method: Trim the electrospun membrane material into a square of 0.5×0.5 cm (this size is used for X-ray photoelectron spectroscopy detection), and set aside. Prepare carboxylated transparent dressings and polyphenol-active protein coating transparent dressings respectively according to the preparation method described in Example 2. After the surface is fully dried, use X-ray photoelectron spectroscopy (model: Perkin Elmer 16PC, with Al target as the anode target) to detect the surface chemical composition, various element compositions and their percentage contents of the two samples.

[0042] Figure 4 For Example 2, a full spectrum of XPS detection of the polyphenol-active protein coating for chronic wound treatment was prepared. From Figure 4 It can be found that compared with the carboxylated surface, an S2p peak appears at 162 - 166 eV for the polyphenol-active protein coating, indicating that the polyphenol-protein composite nanoparticles are effectively modified onto the wound dressing surface.

[0043] Experimental method: The electrospun membrane material was trimmed into a square of 0.5×0.5 cm (this size is for X-ray photoelectron spectroscopy detection) and reserved. According to the preparation method described in Example 2, carboxylated transparent dressings and polyphenol-active protein coating transparent dressings were prepared respectively. After the surface was fully dried, the surface chemical components, various element compositions and their percentage contents of the two samples were detected by X-ray photoelectron spectroscopy (model: Perkin Elmer 16PC, with Al target as the anode target).

[0044] Figure 5 For Example 3, a confocal laser scanning microscopy image of the surface adhesion of macrophages on the polyphenol-active protein coating for chronic wound treatment was prepared. From Figure 3 It can be seen that a large number of macrophages adhere to the surface of the unmodified dressing, and all are in the activated state of extending pseudopods or spreading; while the macrophages adhering to the surface of the polyphenol-active protein coating are in a round resting state, indicating that the constructed polyphenol-active protein coating can effectively inhibit the adhesion and activation of macrophages and regulate the inflammatory response in the chronic wound microenvironment.

[0045] Experimental method: The polyurethane foam wound dressing was trimmed into a square of 1×1 cm (this size is for placing the sample in a 24-well cell culture plate). According to the preparation method described in Example 3, a polyphenol-active protein coating was deposited on the surface of the polyurethane foam wound dressing. The modified and unmodified samples were placed in a 24-well plate and sterilized for later use.

[0046] The macrophage cell line (RAW 264.7) cultured to a stable state was scraped off the cell culture flask by mechanical method, and the cell concentration was diluted to 2.5×10 4 cells / mL; subsequently, 1 mL of the macrophage dilution was added to the sample well plate. When cultured for 3 days, the samples were taken out, and the sample surface was appropriately washed 3 times with PBS solution; the macrophages adhering to the sample surface were stained with rhodamine-labeled phalloidin, and then observed and photographed with a super-high-resolution confocal laser scanning microscope (model: LSM880 Airyscan with STEDYCON) for the surface-adhered macrophages.

[0047] Figure 6For Example 4, the antibacterial rate of the polyphenol-active protein coating for chronic wound treatment was prepared. Starting from Figure 6 It can be found that the antibacterial rates of the gauze and the carboxylated surface against Staphylococcus aureus and Pseudomonas aeruginosa are both less than 50%, and they do not have antibacterial effects; while the antibacterial rate of the polyphenol-active protein coating against both bacteria reaches 99.9%, indicating that the polyphenol-active protein coating can effectively kill bacteria and has excellent antibacterial properties.

[0048] Experimental method: Trim the gauze into a 1×1 cm square (this size is used to place the sample in a 24-well cell culture plate). According to the preparation method described in Example 4, prepare a carboxylated surface and a polyphenol-active protein coating on the surface of the gauze respectively. Place the unmodified gauze, carboxylated gauze, and polyphenol-active protein coating in a 24-well plate and perform aseptic treatment for later use.

[0049] Common strains of chronic wound infections, such as Staphylococcus aureus (S. aureus, ATCC 25923) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853), were used to evaluate the anti-infection performance of different dressings. Dilute the bacteria with PBS solution to a concentration of 2×10 7 CFU / mL; add 1 mL of the bacterial suspension to all sample wells and completely immerse the samples. After incubating in a 37°C incubator for 8 hours, use the microbial viability detection kit WST®-8 (Dojindo LM439, Japan) to evaluate the antibacterial rate of different dressings.

[0050] As described above, this is only the specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A preparation method of a polyphenol-active protein coating for chronic wound treatment, characterized in that The preparation method comprises the following steps: (1) Dissolve dopamine, a carboxyl compound, and an oxidant in an acidic buffer solution respectively to obtain a dopamine solution, a carboxyl compound solution, and an oxidant solution; (2) Mix the dopamine solution, the carboxyl compound solution, and the oxidant solution to obtain a mixed solution; Then completely immerse the wound dressing base material in the mixed solution for reaction and thoroughly wash it to obtain a base material with negatively charged carboxyl groups on the surface; (3) Dissolve a water-insoluble polyphenol and a protein in a PBS solution containing DMSO respectively to obtain a polyphenol solution and a protein solution. Under a magnetic stirring environment, drop the polyphenol solution into the protein solution and continuously stir for reaction to obtain a suspension of polyphenol-protein composite nanoparticles; (4) Immerse the base material with negatively charged carboxyl groups on the surface in the suspension of polyphenol-protein composite nanoparticles for reaction and thoroughly wash it to obtain the polyphenol-active protein coating for treating chronic wounds.

2. The method for preparing the polyphenol-active protein coating for treating chronic wounds according to claim 1, characterized in that: In step (1), the acidic buffer solution is one of acetic acid-acetate buffer solution, 2-(N-morpholino)ethanesulfonic acid buffer solution, glycine-hydrochloric acid buffer solution, phthalic acid-hydrochloric acid buffer solution, potassium hydrogen phthalate-sodium hydroxide buffer solution, disodium hydrogen phosphate-citric acid buffer solution, citric acid-sodium hydroxide-hydrochloric acid buffer solution, and citric acid-sodium citrate buffer solution.

3. The preparation method of the polyphenol-active protein coating for chronic wound treatment according to claim 1, characterized in that, In step (1), the carboxyl compound is one or a combination of two or more of lactic acid, malic acid, tartaric acid, glycine, glutamic acid, α-ketoglutaric acid, vitamin C, acetylsalicylic acid, oxalic acid, phthalic acid, and salicylic acid.

4. The preparation method of the polyphenol-active protein coating for chronic wound treatment according to claim 1, characterized in that, In step (1), the oxidant is one or a combination of two or more of hydrogen peroxide, ammonium persulfate, concentrated nitric acid, sodium periodate, potassium permanganate, and potassium dichromate.

5. The preparation method of the polyphenol-active protein coating for chronic wound treatment according to claim 1, characterized in that, In step (2), the wound dressing base material is one of gauze, cotton pad, transparent dressing, electrospun membrane, chitosan dressing, and polyurethane foam.

6. The method for preparing the polyphenol-active protein coating for treating chronic wounds according to claim 1, characterized in that: In step (3), the water-insoluble polyphenol is one or a combination of two or more of catechin, catechin-3-galucoside, catechin-4'-galucoside, luteolin, quercetin flavanone, hesperetin flavanone, soy isoflavone, flavanone, liquiritigenin, oleuropein, hydroxytyrosol, caffeophenol ester, procyanidin, quercetin, anthocyanin, magnolol, and honokiol.

7. The preparation method of the polyphenol-active protein coating for chronic wound treatment according to claim 1, characterized in that, In step (3), the protein is one or a combination of two or more of growth factor proteins, structural proteins and extracellular matrix-related proteins, antimicrobial peptides and immunomodulatory peptides, synthetic peptides and biomimetic peptides; and / or, the growth factor proteins are one or a combination of two or more of epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, and transforming growth factor-β; and / or, the structural proteins and extracellular matrix-related proteins are one or a combination of two or more of collagen, fibronectin, laminin, albumin, fibrinogen, type I collagen, type II collagen, type III collagen, type IV collagen, and lysozyme; And / or, the antimicrobial peptide and the immunomodulatory peptide are one or a combination of two or more of LL-37, α-defensin, β-defensin, Tβ4, lactoferrin, QQKFQFQFEQQ peptide, and KLAKLAK peptide; And / or, the synthetic polypeptide and the biomimetic peptide are one or a combination of two or more of RGD peptide, copper peptide GHK-Cu, and lipopeptide PXL01.

8. The preparation method of the polyphenol-active protein coating for chronic wound treatment according to claim 1, characterized in that, In step (1), the pH value of the acidic buffer solution is 2 to 6.5; And / or, in step (1), the mass concentrations of the dopamine solution, the carboxyl compound solution, and the oxidant solution are 0.5 to 50 mg / mL, 0.5 to 20 mg / mL, and 0.5 to 20 mg / mL, respectively; And / or, in step (3), the volume concentration of DMSO in the PBS solution containing DMSO is 0.5 to 10%; And / or, in step (3), the mass concentrations of the water-insoluble polyphenol solution and the protein solution are 0.1 to 10 mg / mL and 1 to 100 mg / mL, respectively.

9. The preparation method of the polyphenol-active protein coating for chronic wound treatment according to claim 1, characterized in that, In step (2), the reaction time is 0.5 to 24 h, and the reaction temperature is 15 to 40 °C; And / or, in step (3), the reaction time is 1 to 48 h, the stirring speed is 600 to 800 rpm, and the reaction temperature is 10 to 37 °C; And / or, in step (4), the reaction time is 0.5 to 48 h, and the reaction temperature is 10 to 37 °C.

10. A polyphenol-active protein coating for treating chronic wounds obtained by the preparation method according to any one of claims 1 to 9.

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