A polyphenol-active protein coating for treating chronic wounds and its preparation method

Abstract

The present invention belongs to the field of biomaterial technology, and specifically relates to a polyphenol-active protein coating for the treatment of chronic wounds and a preparation method thereof. The preparation method is to first utilize the non-material dependence of the polydopamine coating to form a carboxylated negatively charged surface on the surface of different dressings; then utilize electrostatic interaction to fix polyphenol-protein composite nanoparticles rich in multiple active sites and carrying positive charges on the surface of the dressing. The preparation method can directly construct a coating on the surface of different base dressing materials such as gauze, cotton pads, transparent dressings, electrospinning membranes, chitosan dressings, polyurethane foams, etc., and selectively adsorb different polyphenols and proteins on the surface of the dressing according to the different stages of wound healing, so that the surface has specific or broad-spectrum biological functions, which better meet the biosafety and biofunctionality of chronic wound dressings.

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] Wound dressings, as an effective means of managing diabetic wounds, can enhance therapeutic efficacy by protecting the wound surface, reducing infection, and maintaining a moist environment. Currently, gauze, cotton pads, transparent dressings, silver ion dressings, and silicone dressings are widely used in clinical practice. However, they still present challenges such as easy adhesion to wounds, resulting in secondary damage, poor breathability, lack of water absorption, potential for drug resistance or cytotoxicity with long-term use, and high cost. Furthermore, single basic dressings face multiple challenges in regulating the complex microenvironment of diabetic wounds. Therefore, functionalizing basic dressings through surface modification techniques to precisely adapt them to the multidimensional needs of diabetic wounds holds significant research value. Biomimetic strategies from nature provide important inspiration for material design. Research has shown that naturally occurring polyphenol-protein complexes in plants often possess antimicrobial, antioxidant, anti-inflammatory, and structural strengthening properties, which enable plants to defend against biological threats and promote wound healing. Surface engineering strategies have been widely studied as an effective means of imparting multifunctional activity to the surfaces of biomedical materials and devices. Therefore, the preparation of polyphenol-protein coatings on wound dressing substrates through surface modification techniques is of great significance for promoting wound healing. However, current methods for preparing polyphenol-protein surfaces primarily include: 1) layer-by-layer self-assembly—the alternating deposition of oppositely charged polyphenols and protein molecules utilizes electrostatic attraction to build the coating layer by layer; 2) covalent crosslinking—the formation of stable coatings using chemical crosslinkers (such as genipin and glutaraldehyde) or oxidative coupling reactions (polyphenols oxidized to quinones and then reacted with proteins); 3) physical adsorption after blending—the direct coating of polyphenol and protein solutions onto the surface to form a film; 4) electrochemical deposition; and 5) microfluidics / 3D printing. These methods can form polyphenol-protein coatings on various substrates as needed. However, they also face common challenges, such as insufficient stability, time-consuming processes, loss of activity, potential toxicity, high environmental sensitivity, low loading capacity, high equipment costs, and low technological maturity, which significantly limit the large-scale or commercial application of polyphenol-protein coatings in chronic wound dressings.

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

[0005] To address the challenges of the existing technology, the present invention provides a polyphenol-active protein coating for chronic wound treatment and its preparation method. This preparation method allows for the direct construction of a polyphenol-protein coating on the surface of various wound dressing substrates (such as gauze, cotton pads, transparent dressings, electrospun membranes, chitosan dressings, and polyurethane foam). Based on the wound's healing status, the coating selectively adsorbs different polyphenols and proteins onto the surface, imparting specific or broad-spectrum biological functions to the surface, thereby better meeting the biosafety and biofunctionality requirements of the wound dressing.

[0006] The present invention provides a method for preparing a polyphenol-active protein coating for treating chronic wounds, the method comprising the following steps:

[0007] (1) dissolving dopamine, a carboxyl compound, and an oxidant in an acidic buffer solution to obtain a dopamine solution, a carboxyl compound solution, and an oxidant solution;

[0008] (2) mixing the dopamine solution, the carboxyl compound solution and the oxidant solution to obtain a mixed solution; then completely immersing the wound dressing base material in the mixed solution to react and fully wash the wound dressing base material to obtain a base material with a negatively charged surface carboxyl group;

[0009] (3) dissolving the water-insoluble polyphenol and protein in a PBS solution containing DMSO, respectively, to obtain a polyphenol solution and a protein solution; and adding the polyphenol solution dropwise into the protein solution under magnetic stirring, and continuously stirring to react, to obtain a polyphenol-protein composite nanoparticle suspension;

[0010] (4) Immersing the surface carboxylated negatively charged substrate material in the polyphenol-protein composite nanoparticle suspension for reaction and thorough washing to obtain the polyphenol-active protein coating for chronic wound treatment.

[0011] In order to facilitate understanding of the present invention, the reaction principle of the present invention is explained:

[0012] In the preparation method of the present invention, dopamine and a carboxyl compound are first oxidized, crosslinked, and polymerized under the action of an oxidant to form a carboxylated negatively charged surface on the surface of wound dressing substrates of different shapes and materials. Subsequently, a water-insoluble polyphenol compound and protein are dissolved separately in a PBS solution containing DMSO. The polyphenol compound and protein achieve multi-scale intermolecular bonding under the single or dynamic synergistic effects of hydrogen bonding, hydrophobic interactions, π-π stacking effects, electrostatic attraction, and van der Waals forces, ultimately self-assembling to form structurally stable polyphenol-protein composite nanoparticles. These polyphenol-protein composite nanoparticles are rich in multiple active sites and carry a positive charge. When they come into contact with the negatively charged carboxylated substrate surface, they are anchored in situ through an electrostatically driven self-assembly strategy, thereby forming a stable super-hydrophilic polyphenol-active protein coating on the surface of the chronic wound dressing. Notably, the formation and immobilization of polyphenol-protein composite nanoparticles are primarily achieved through non-covalent bonding. This mild bonding strategy fully preserves the polyphenol's phenolic hydroxyl active site and the protein's native conformation. Based on the precise synergy of functional modules (the polyphenol's triple antioxidant, antimicrobial, and immune-modulating effects combined with the protein's dual antibacterial and healing-promoting effects), the system achieves multi-level integration of active ingredients, ultimately constructing a chronic wound repair dressing system with a polyphenol-protein multi-level biological synergistic mechanism. Furthermore, because the binding and immobilization of polyphenols and proteins are primarily achieved through coating electrostatic attraction, hydrogen bonding networks, and hydrophobic interactions, the polyphenol-active protein coating on the dressing surface is pH-responsive. This accelerates the release of polyphenols and active proteins in the weakly acidic microenvironment (pH ≈ 5.5-6.5) created by bacterial infection, thereby timely regulating the stability of the microenvironment.

[0013] Preferably, in step (1), the acidic buffer is one of acetic acid-acetate buffer, 2-(N-morpholinoline)ethanesulfonic acid buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, potassium hydrogen phthalate-sodium hydroxide buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, and citric acid-sodium citrate buffer.

[0014] 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.

[0015] 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.

[0016] Preferably, in step (2), the base material of the wound dressing is one of gauze, cotton pad, transparent dressing, electrospinning membrane, chitosan dressing, and polyurethane foam.

[0017] Preferably, in step (3), the water-insoluble polyphenol is one or a combination of two or more of catechin, catechin-3-galucoside, catechin-4'-galucoside, tartrazine, quercetin flavanone, hesperetin flavanone, soy isoflavone, flavanone, glycyrrhizin, olive phenol, hydroxytyrosol, caffeol ester, proanthocyanidin, quercetin, anthocyanidin, magnolol, and magnolol.

[0018] 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 polypeptides and biomimetic peptides;

[0019] And / or, the growth factor protein is 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;

[0020] And / or, the structural protein and extracellular matrix-related protein 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;

[0021] And / or, the antimicrobial peptide and 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;

[0022] And / or, the synthetic polypeptide and biomimetic peptide are one or a combination of two or more of RGD peptide, copper peptide GHK-Cu, and lipopeptide PXL01.

[0023] Preferably, in step (1), the pH value of the acidic buffer solution is 2-6.5;

[0024] and / or, in step (1), the mass concentrations of the dopamine solution, the carboxyl compound solution, and the oxidant solution are 0.5-50 mg / mL, 0.5-20 mg / mL, and 0.5-20 mg / mL, respectively;

[0025] And / or, in step (3), the volume concentration of DMSO in the PBS solution containing DMSO is 0.5-10%;

[0026] and / or, in step (3), the mass concentrations of the water-insoluble polyphenol solution and the protein solution are 0.1-10 mg / mL and 1-100 mg / mL, respectively;

[0027] Preferably, in step (2), the reaction time is 0.5-24 h, and the reaction temperature is 15-40°C;

[0028] And / or, in step (3), the reaction time is 1-48 h, the stirring speed is 600-800 rpm, and the reaction temperature is 10-37°C;

[0029] And / or, in step (4), the reaction time is 0.5-48 h, and the reaction temperature is 10-37°C.

[0030] Based on the same technical concept, another object of the present invention is to provide a polyphenol-active protein coating obtained by the above preparation method for treating chronic wounds.

[0031] The beneficial effects of the present invention are:

[0032] 1. Active protein coatings have excellent biocompatibility and biofunctionality and are widely used in the field of biomaterial surface modification. Currently, proteins are modified to the surface of materials mainly through physical adsorption, chemical cross-linking and layer-by-layer self-assembly. However, there is still the problem of difficulty in achieving both protein activity and stability. The preparation method described in the present invention first prepares a carboxylated negatively charged surface with excellent wettability and micro-nano topological structure; then, the positively charged polyphenol-protein composite nanoparticles are stably fixed to the dressing surface through electrostatic adsorption. During the entire process, the binding and fixation of proteins are mainly based on non-covalent bonds, which effectively preserves the structure and function of the protein.

[0033] 2. Polyphenols are naturally active small molecules with excellent biological functions such as antioxidant, antibacterial, and inflammation-modulating properties. However, the phenolic hydroxyl groups of polyphenols are easily oxidized, which reduces their antioxidant and inflammation-modulating properties. The preparation method described in the present invention non-covalently binds polyphenols to proteins, effectively slowing the oxidation of phenolic hydroxyl groups and preserving the biological functions of polyphenols.

[0034] 3. The microenvironment of chronic wounds is hindered by factors such as excessive oxidative stress, inflammatory disorders, metabolic abnormalities, microbial infection, and cellular dysfunction, making it difficult to heal. However, single functional proteins or active molecules cannot effectively address these adverse microenvironmental factors. The preparation method described in this invention utilizes precise synergy of functional modules (the triple antioxidant, antimicrobial, and immunomodulatory effects of polyphenols combined with the dual antibacterial and healing-promoting effects of proteins), systematically achieving a multi-level integration of the active ingredients polyphenols and proteins.

[0035] 4. Chronic wounds are prone to bacterial infection, creating a weakly acidic microenvironment (pH ≈ 5.5-6.5). This can hinder tissue repair through various mechanisms, such as inhibiting the proliferation and migration of repair cells, inhibiting enzyme activity, and exacerbating inflammatory responses. Therefore, timely prevention of bacterial infection is crucial for the healing of chronic wounds. The polyphenol-protein binding and fixation described in the preparation method of the present invention are primarily achieved through electrostatic attraction, hydrogen bonding networks, and hydrophobic interactions in the coating. Therefore, the polyphenol-active protein coating on the dressing surface is pH-responsive. This accelerates the release of polyphenols and active proteins in the weakly acidic microenvironment created by bacterial infection, effectively killing bacteria and regulating the stability of the microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0037] Figure 1 Zeta potential diagram of the carboxylated surface and polyphenol-protein composite nanoparticle suspension in the polyphenol-active protein coating prepared for chronic wound treatment in Example 1.

[0038] Figure 2 This is a scanning electron micrograph of the polyphenol-active protein coating for treating chronic wounds prepared in Example 1.

[0039] Figure 3 This is a graph showing the element content results of XPS testing of the polyphenol-active protein coating for chronic wound treatment prepared in Example 2.

[0040] Figure 4 This is the full spectrum of XPS detection of the polyphenol-active protein coating prepared for chronic wound treatment in Example 2.

[0041] Figure 5 This is a laser confocal image of macrophage skeleton staining on the surface of the polyphenol-active protein coating for chronic wound treatment prepared in Example 3.

[0042] Figure 6 The antibacterial rate of the polyphenol-active protein coating for treating chronic wounds prepared in Example 4 is shown. DETAILED DESCRIPTION

[0043] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0044] Example 1

[0045] This embodiment provides a method for preparing a polyphenol-active protein coating for wound treatment, the preparation method comprising:

[0046] (1) Prepare acetic acid-acetate buffer at pH 3, dissolve dopamine, malic acid, and potassium permanganate oxidant to form dopamine solution, malic acid solution, and potassium permanganate solution with mass concentrations of 6 mg / mL, 6 mg / mL, and 6 mg / mL, respectively;

[0047] (2) Mixing equal volumes of the dopamine solution, malic acid solution, and potassium permanganate solution prepared in (1), completely immersing the transparent film wound dressing in the mixture, reacting at a constant temperature of 25°C for 2 hours, and thoroughly washing to obtain a transparent film wound dressing with a negatively charged surface carboxyl group;

[0048] (3) Magnolia officinalis and lysozyme were dissolved in PBS solution containing DMSO at a volume concentration of 1% to obtain magnolia officinalis solution and lysozyme solution with mass concentrations of 4 mg / mL and 40 mg / mL, respectively. Under a magnetic stirring environment, an equal volume of magnolia officinalis solution was added dropwise to the lysozyme solution. The mixture was stirred at a speed of 600 rpm for 5 hours until a stable and uniform suspension of magnolia officinalis-lysozyme composite nanoparticles was formed for later use.

[0049] (4) The surface carboxylated negatively charged transparent film wound dressing prepared in (2) is immersed in the magnolol-lysozyme composite nanoparticle suspension prepared in (3), and the mixture is reacted at a constant temperature of 25°C for 10 hours, and then thoroughly washed to obtain the magnolol-lysozyme coating for chronic wound treatment.

[0050] Example 2

[0051] This embodiment provides a method for preparing a polyphenol-active protein coating for treating chronic wounds, wherein the preparation method comprises:

[0052] (1) Prepare glycine-hydrochloric acid buffer with a pH of 5, dissolve dopamine, glycine, and sodium periodate oxidant to form dopamine solution, glycine solution, and sodium periodate solution with mass concentrations of 9 mg / mL, 6 mg / mL, and 9 mg / mL, respectively;

[0053] (2) Mixing equal volumes of the dopamine solution, glycine solution, and sodium periodate solution prepared in (1), completely immersing the electrospun membrane wound dressing in the mixture, reacting at a constant temperature of 20°C for 5 hours, and thoroughly washing to obtain an electrospun membrane wound dressing with a negatively charged surface carboxyl group;

[0054] (3) Dissolve quercetin and IV collagen in a PBS solution containing DMSO at a volume concentration of 5% to obtain a quercetin solution and IV collagen solution with a mass concentration of 1 mg / mL and 10 mg / mL, respectively. Add an equal volume of quercetin solution dropwise into the IV collagen solution under magnetic stirring. Stir the mixture at a speed of 650 rpm for 8 hours until a stable and uniform quercetin-IV collagen composite nanoparticle suspension is formed for later use.

[0055] (4) The surface carboxylated negatively charged electrospun membrane wound dressing prepared in (2) is immersed in the quercetin-IV collagen composite nanoparticle suspension prepared in (3), and the mixture is reacted at a constant temperature of 20°C for 20 hours, and then thoroughly washed to obtain a quercetin-IV collagen coating for chronic wound treatment.

[0056] Example 3

[0057] This embodiment provides a method for preparing a polyphenol-active protein coating for treating chronic wounds, wherein the preparation method comprises:

[0058] (1) Prepare sodium hydrogen phosphate-citric acid buffer with a pH of 5.5, dissolve dopamine, salicylic acid, and ammonium persulfate oxidant to form dopamine solution, salicylic acid solution, and ammonium persulfate solution with mass concentrations of 3 mg / mL, 6 mg / mL, and 6 mg / mL, respectively;

[0059] (2) mixing equal volumes of the dopamine solution, salicylic acid solution, and ammonium persulfate solution prepared in (1), completely immersing the polyurethane foam wound dressing in the mixture, reacting at a constant temperature of 30°C for 2 hours, and thoroughly washing to obtain a polyurethane foam wound dressing with a negatively charged surface carboxyl group;

[0060] (3) Dissolve proanthocyanidins and antimicrobial peptide QQKFQFQFEQQ in PBS solution containing DMSO at a volume concentration of 1.5% to obtain proanthocyanidin solution and antimicrobial peptide QQKFQFQFEQQ solution with mass concentrations of 4 mg / mL and 20 mg / mL, respectively. Add equal volumes of proanthocyanidin solution dropwise into antimicrobial peptide QQKFQFQFEQQ solution under magnetic stirring, and stir the mixture at a speed of 700 rpm for 12 hours until a stable and uniform proanthocyanidin-antimicrobial peptide composite nanoparticle suspension is formed for later use.

[0061] (4) The surface carboxylated negatively charged polyurethane foam wound dressing prepared in (2) is immersed in the suspension of proanthocyanidin-antimicrobial peptide composite nanoparticles prepared in (3), and the mixture is reacted at a constant temperature of 30°C for 2 hours, and then thoroughly washed to obtain a proanthocyanidin-antimicrobial peptide coating for chronic wound treatment.

[0062] Example 4

[0063] This embodiment provides a method for preparing a polyphenol-active protein coating for treating chronic wounds, wherein the preparation method comprises:

[0064] (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 dopamine solution, α-ketoglutaric acid solution, and potassium dichromate solution with mass concentrations of 12 mg / mL, 12 mg / mL, and 6 mg / mL, respectively;

[0065] (2) Mixing equal volumes of the dopamine solution, α-ketoglutaric acid solution, and potassium dichromate solution prepared in (1), completely immersing the gauze wound dressing in the mixture, reacting at a constant temperature of 20°C for 12 hours, and thoroughly washing to obtain a transparent film wound dressing with a negatively charged surface carboxyl group;

[0066] (3) Dissolve honokiol and fibroblast growth factor in PBS solution containing DMSO at a volume concentration of 1% to obtain honokiol solution and fibroblast growth factor solution with mass concentrations of 6 mg / mL and 100 ng / mL, respectively. Add equal volumes of honokiol solution dropwise into the fibroblast growth factor solution under magnetic stirring. Stir the mixture at a speed of 600 rpm for 1 hour until a stable and uniform suspension of honokiol-fibroblast growth factor composite nanoparticles is formed for later use.

[0067] (4) The surface carboxylated negatively charged transparent film wound dressing prepared in (2) is immersed in the suspension of magnolia officinalis-fibroblast growth factor composite nanoparticles prepared in (3), and the mixture is reacted at a constant temperature of 20°C for 15 hours, and then thoroughly washed to obtain the magnolia officinalis-fibroblast growth factor coating for chronic wound treatment.

[0068] Verification Example

[0069] Figure 1 The zeta potential of the carboxylated surface and polyphenol-protein composite nanoparticle suspension in the polyphenol-active protein coating prepared for chronic wound treatment in Example 1 was obtained. Figure 1It can be seen that the Zeta potential of the prepared carboxylation surface and the polyphenol-protein composite nanoparticle suspension are approximately -43 mV and 28 mV, respectively, indicating that the carboxylation surface mainly fixes the polyphenol-protein composite nanoparticles on the dressing surface through electrostatic interaction, effectively retaining the biological activity of polyphenols and proteins.

[0070] Experimental method: The transparent film dressing was trimmed into a 1×2 cm rectangle (the sample size requirement of the solid surface zeta potential tester) and set aside. According to steps (1) and (2) of the preparation method described in Example 1, a carboxylation surface was deposited on the surface of the 1×2 cm rectangular transparent dressing. Subsequently, a polyphenol-protein composite nanoparticle suspension was prepared according to step (3) of the preparation method described in Example 1. Finally, the charge values ​​of the carboxylation surface and the polyphenol-protein composite nanoparticle suspension were measured using a solid surface zeta potential tester (model: SurPASS) and a nanoparticle sizer and zeta potential analyzer (model: Zeta sizer Nano ZS), respectively.

[0071] Figure 2 The SEM surface morphology of the polyphenol-active protein coating prepared for chronic wound treatment in Example 1 is as follows: Figure 2 As shown in the figure, the surface of the carboxylated substrate presents a micro-nano composite topological morphology. After modification with polyphenol-protein composite nanoparticles, the substrate surface is evenly coated, confirming that the polyphenol-protein composite nanoparticles are successfully grafted onto the carboxylated surface, realizing the controllable construction of the active protein coating.

[0072] Experimental Methods: Transparent dressings were trimmed into 0.8 × 0.8 cm squares (this size was used for field emission scanning electron microscopy), and the front and back surfaces of the samples were labeled for later use. Following the preparation method described in Example 1, a carboxylated transparent dressing and a polyphenol-active protein-coated transparent dressing were prepared. The prepared surfaces were then air-dried and sprayed with gold (spraying time 90 seconds). The surface morphologies of the different samples were then observed using a field emission scanning electron microscope (S-4800).

[0073] Figure 3 The element content results of the XPS test of the polyphenol-active protein coating for chronic wound treatment prepared in Example 2 are as follows: Figure 3 It can be seen that compared with the chemical elements on the carboxylated surface, the polyphenol-active protein coating contains 0.94% S element, and the N element in the coating is significantly increased, while the C element content is decreased. The S element is a unique element that distinguishes protein from polyphenol, further indicating that the protein was successfully grafted to the carboxylated surface.

[0074] Experimental Method: The electrospun membrane material was trimmed into 0.5 × 0.5 cm squares (this size was used for X-ray photoelectron spectroscopy) and set aside. A carboxylated transparent dressing and a polyphenol-active protein-coated transparent dressing were prepared according to the method described in Example 2. After the surfaces were fully dried, the surface chemical composition, elemental composition, and percentages of the two samples were determined using X-ray photoelectron spectroscopy (Perkin Elmer 16PC, Al target as the anode).

[0075] Figure 4 The full spectrum of XPS detection of polyphenol-active protein coating for chronic wound treatment prepared in Example 2 is as follows: Figure 4 It can be found that compared with the carboxylated surface, the polyphenol-active protein coating showed an S2p peak at 162~166 eV, indicating that the polyphenol-protein composite nanoparticles were effectively modified to the wound dressing surface.

[0076] Experimental Method: The electrospun membrane material was trimmed into 0.5 × 0.5 cm squares (this size was used for X-ray photoelectron spectroscopy) and set aside. A carboxylated transparent dressing and a polyphenol-active protein-coated transparent dressing were prepared according to the method described in Example 2. After the surfaces were fully dried, the surface chemical composition, elemental composition, and percentages of the two samples were determined using X-ray photoelectron spectroscopy (Perkin Elmer 16PC, Al target as the anode).

[0077] Figure 5 Laser confocal microscopy of the morphology of macrophages adhered to the surface of the polyphenol-active protein coating prepared for chronic wound treatment in Example 3. Figure 3 It can be seen that a large number of macrophages adhere to the surface of the unmodified dressing, and they are all in an activated state with pseudopodia extended or spread; while the macrophages adhered 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 of the chronic wound microenvironment.

[0078] Experimental method: The polyurethane foam wound dressing was trimmed into 1×1 cm squares (this size is used to place samples in a 24-well cell culture plate). A polyphenol-active protein coating was deposited on the surface of the polyurethane foam wound dressing according to the preparation method described in Example 3. The modified and unmodified samples were placed in a 24-well plate and sterilized for later use.

[0079] The macrophage cell line (RAW 264.7) that had been cultured to a stable state was mechanically scraped from the cell culture flask and the cell concentration was diluted to 2.5×10 4cells / mL; then, 1 mL of macrophage dilution was added to the sample wells. After 3 days of culture, the samples were removed and the surface was washed three times with PBS. Macrophages adhered to the sample surface were stained with rhodamine-labeled phalloidin and observed and photographed using an ultra-high-resolution laser confocal microscope (LSM880 Airyscan with STEDYCON).

[0080] Figure 6 The antibacterial rate of the polyphenol-active protein coating prepared for chronic wound treatment in Example 4 is as follows: Figure 6 It can be found that the antibacterial rates of gauze and carboxylated surfaces against Staphylococcus aureus and Pseudomonas aeruginosa are less than 50%, and they have no antibacterial effect; while the antibacterial rates of the polyphenol-active protein coating against both bacteria reached 99.9%, indicating that the polyphenol-active protein coating can effectively kill bacteria and has excellent antibacterial properties.

[0081] Experimental method: Gauze was trimmed into 1 × 1 cm squares (this size is used to place samples in a 24-well cell culture plate). According to the preparation method described in Example 4, the following were prepared on the surface of the gauze: a carboxylated surface and a polyphenol-active protein coating. Unmodified gauze, carboxylated gauze, and polyphenol-active protein coating were placed in a 24-well plate and sterilized for later use.

[0082] The anti-infection performance of different dressings was evaluated using common bacterial strains of chronic wound infection, such as Staphylococcus aureus (S. aureus, ATCC 25923) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 27853). The bacteria were diluted with PBS solution to a concentration of 2×10 7 CFU / mL; 1 mL of bacterial suspension was added to each sample well to completely submerge the sample. After incubation in a 37°C incubator for 8 hours, the antibacterial rate of the different dressings was evaluated using the Microbial Viability Test Kit WST®-8 (Dojindo LM439, Japan).

[0083] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for preparing a polyphenol-active protein coating for treating chronic wounds, characterized in that: The preparation method comprises the following steps: (1) dissolving dopamine, a carboxyl compound, and an oxidant in an acidic buffer solution to obtain a dopamine solution, a carboxyl compound solution, and an oxidant solution; (2) mixing the dopamine solution, the carboxyl compound solution, and the oxidant solution to obtain a mixed solution; Then, the wound dressing base material is completely immersed in the mixed solution to react and is fully washed to obtain a base material with a carboxylated surface and negative charge; (3) dissolving the water-insoluble polyphenol and protein in a PBS solution containing DMSO, respectively, to obtain a polyphenol solution and a protein solution; and adding the polyphenol solution dropwise into the protein solution under magnetic stirring, and continuously stirring to react, to obtain a polyphenol-protein composite nanoparticle suspension; (4) Immersing the surface carboxylated negatively charged substrate material in the polyphenol-protein composite nanoparticle suspension for reaction and thorough washing to obtain the polyphenol-active protein coating for chronic wound treatment.

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 is one of acetic acid-acetate buffer, 2-(N-morpholinoline)ethanesulfonic acid buffer, glycine-hydrochloric acid buffer, phthalic acid-hydrochloric acid buffer, potassium hydrogen phthalate-sodium hydroxide buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, and citric acid-sodium citrate buffer.

3. The method for preparing the polyphenol-active protein coating for treating chronic wounds 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 method for preparing the polyphenol-active protein coating for treating chronic wounds 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 method for preparing the polyphenol-active protein coating for treating chronic wounds according to claim 1, characterized in that: In step (2), the base material of the wound dressing is one of gauze, cotton pad, transparent dressing, electrospinning 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, tartrazine, quercetin flavanone, hesperetin flavanone, soy isoflavone, flavanone, glycyrrhizin, olive phenol, hydroxytyrosol, caffeol ester, proanthocyanidin, quercetin, anthocyanidin, magnolol, and magnolol.

7. The method for preparing the polyphenol-active protein coating for treating chronic wounds 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 protein is 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 protein and extracellular matrix-related protein 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 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 biomimetic peptide are one or a combination of two or more of RGD peptide, copper peptide GHK-Cu, and lipopeptide PXL01.

8. The method for preparing the polyphenol-active protein coating for treating chronic wounds 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 0.5-50 mg / mL, 0.5-20 mg / mL and 0.5-20 mg / mL, respectively; And / or, in step (3), the volume concentration of DMSO in the PBS solution containing DMSO is 0.5-10%; And / or, in step (3), the mass concentrations of the water-insoluble polyphenol solution and the protein solution are 0.1-10 mg / mL and 1-100 mg / mL, respectively.

9. The method for preparing the polyphenol-active protein coating for treating chronic wounds according to claim 1, characterized in that: In step (2), the reaction time is 0.5-24 h, and the reaction temperature is 15-40°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 0.5-48 h, and the reaction temperature is 10-37°C.

10. The 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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