A tannin hybrid naringin-copper ion nanonetwork liquid dressing and a preparation method and application thereof

Abstract

The application provides a preparation method of a tannin hybrid naringin-copper ion nanometer network liquid dressing, which comprises the following steps: naringin is mixed with a copper salt solution for reaction, the precipitate is collected by centrifugation, then tannin acid solution is added, and ultrasonic stirring is carried out to mix, so that the target liquid dressing is obtained. The application firstly constructs a naringin-copper (II) primary network framework through metal coordination driving, and then forms a metal-polyphenol semi-interpenetrating supramolecular nanometer network structure through tannin acid ligand exchange, π-π stacking interlocking and hydrogen bond "suture" interface. By referring to the strong adhesion characteristics of catechol groups in mussel adhesion proteins, tannin acid is stably combined with the nanometer network surface, bacterial cell wall proteins and wound collagen through its catechol structure, so that the stability of the liquid dressing is enhanced, and the liquid dressing is endowed with the ability to capture bacteria and wound adhesion. In addition, tannin acid and naringin are both natural compounds, have good biocompatibility and degradability, and can reduce the irritation of the material to biological tissues.

Description

Technical Field

[0001] This invention relates to the field of materials technology, and in particular to a tannin-hybridized naringin-copper ion nanonetwork liquid dressing, its preparation method, and its application. Background Technology

[0002] Anti-infective therapy is one of the core needs in the biomedical field, playing an irreplaceable role in wound repair, implant device protection, and the treatment of infectious diseases. However, traditional anti-infective materials face many insurmountable limitations in clinical applications, severely restricting therapeutic efficacy and the scope of application.

[0003] First, the problem of drug resistance is becoming increasingly serious. The overuse of traditional antibiotics has led to the widespread spread and rapid evolution of multidrug-resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA). More concerning is that bacterial biofilms (bacterial communities encapsulated by an extracellular polymer matrix) further enhance bacterial resistance to antibiotics, reducing antibiotic efficacy by 60%-80%, becoming a key obstacle in the clinical treatment of infections.

[0004] Secondly, biocompatibility and potential toxicity limit the long-term application of traditional antibacterial materials. Some traditional antibacterial materials, such as heavy metal-containing nanoparticles and nanomaterials synthesized with organic solvents, while possessing certain antibacterial activity, may induce adverse effects such as cytotoxicity and inflammatory responses in vivo. For example, while high-concentration silver nanoparticles can exert antibacterial effects, they may damage normal tissue cells, significantly limiting their application in long-term implantation in vivo and wound repair.

[0005] Furthermore, traditional anti-infective materials generally suffer from the limitation of single-function treatment. Clinical anti-infective therapy often needs to simultaneously meet multiple requirements such as sterilization, anti-inflammation, and promotion of tissue healing. However, traditional antibiotics can only achieve a single sterilization function and cannot regulate the inflammatory microenvironment or promote the regeneration of damaged tissues, resulting in slow healing of infected sites, a high incidence of complications, and difficulty in achieving ideal therapeutic effects.

[0006] To address the aforementioned issues, metal-organic framework nanomaterials (MOFs) offer a new direction for supplementing and upgrading traditional antibiotics. They possess advantages such as low cost, high chemical stability, large-scale production capability, and tunable functionality. Their catalytic activity can be optimized through precise control of size, morphology, and composition, achieving synergistic effects of highly efficient antibacterial and anti-inflammatory properties. This provides new insights for the development of multifunctional anti-infective materials.

[0007] However, existing MOFs still have significant shortcomings: First, they lack stability. Many MOFs are prone to hydrolysis in water or humid environments, leading to framework collapse and affecting the biosafety of the material. Their crystal structures are usually brittle, easily crushed or structurally destroyed under mechanical stress such as pressure, friction, or vibration, affecting their durability in practical devices. Second, they have low functional integration. The functional design of MOFs often focuses on a single area (such as gas adsorption or catalysis), lacking multifunctional synergistic integration, making it difficult to match the composite needs of clinical anti-infection and environmental remediation scenarios. Although the combination of MOFs with noble metal nanoparticles, polymers, and other materials can improve performance, problems such as weak interfacial bonding and uneven component distribution prevent the synergistic effect from being fully realized. Third, the preparation process has limitations. Traditional synthesis methods or processes are complex and costly, or difficult to achieve large-scale mass production, limiting their industrial application and clinical translation.

[0008] Therefore, developing a novel anti-infective material with excellent biocompatibility, high functional integration, simple preparation process, and scalable production, in order to overcome the many shortcomings of traditional anti-infective materials and existing MOFs and meet the multiple needs of clinical anti-infective therapy, has become an urgent technical problem to be solved in this field. Summary of the Invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide a tannic acid hybrid naringin-copper ion nanonetwork liquid dressing. The material provided by the present invention has a nanonetwork structure, which has significant advantages over conventional spherical nanoparticles in the fields of antibacterial and wound repair: as a "highly efficient trap" for bacterial capture, the three-dimensional network structure provides bacteria with a large number of surface binding sites, and with the strong adhesion of tannic acid and catechol, the bacterial capture ability is further enhanced and the movement of bacteria is restricted, forming a high-concentration antibacterial environment in the local wound, effectively improving the antibacterial effect; through mechanical interlocking, the material is tightly adhered to the wound surface, which not only continuously releases antibacterial components, but also promotes tissue regeneration, significantly promotes wound healing, and improves the quality of healing. In summary, the network structure nanomaterial, through its unique three-dimensional network design, precise targeted adhesion mechanism, and multifunctional integrated characteristics, breaks through the limitations of spherical nanoparticles in the fields of antibacterial and wound repair, and provides a better solution for related fields.

[0010] This invention provides a method for preparing a tannin-hybridized naringin-copper ion nanonetwork liquid dressing, comprising:

[0011] Naringin and copper salt are first mixed and reacted, centrifuged, and the supernatant is discarded. Then, tannic acid is added and the mixture is ultrasonically stirred to obtain the final product.

[0012] In some embodiments, the molar ratio of the naringin and copper salt is 1:8.

[0013] The concentration of copper ions in the copper salt is 2~3 mg / mL.

[0014] In some embodiments, the copper salt includes copper sulfate, copper nitrate, or copper chloride.

[0015] In some embodiments, the concentration of the tannic acid is 3-5 mg / mL, the pH value is 7.2, and the amount added is 1 mL.

[0016] In some embodiments, the reaction of naringin and copper salt is an ultrasonic reaction, and the ultrasonic time is 5 min; the centrifugation is 14000 rpm for 20 min.

[0017] In some embodiments, the ultrasonication time after adding tannic acid is 10 min, the stirring time and speed are 400 rpm, and the time is 4 h; the centrifugation is 14000 rpm for 30 min; and the suspension is resuspended in ddH2O after centrifugation.

[0018] This invention provides a tannic acid hybrid naringin-copper ion nanonetwork liquid dressing, which is prepared by any of the preparation methods described in the above technical solutions.

[0019] This invention provides the application of the tannic acid hybrid naringin-copper ion nanonetwork liquid dressing described above in the preparation of products that promote wound healing and / or improve healing quality.

[0020] The wound-healing-promoting properties include one or more of the following: anti-inflammatory, antioxidant, and antibacterial.

[0021] Improving healing quality includes promoting tissue regeneration and angiogenesis.

[0022] This invention provides a product that promotes wound healing and / or improves the quality of healing, including the naringin-copper ion nanonetwork liquid dressing described in the above technical solution.

[0023] The product described in this invention can be prepared in various dosage forms such as hydrogels and microneedles, allowing for flexible selection based on wound type, healing stage, and application scenario. The hydrogel formulation can adhere to irregular wound surfaces, maintain a moist environment, and exhibits excellent adhesion, preventing easy detachment. The microneedle formulation delivers active ingredients to deeper layers of the wound through minimally invasive delivery, enhancing efficacy and reducing component loss.

[0024] Compared with existing technologies, this invention provides a method for preparing a tannic acid-hybridized naringin-copper ion nanonetwork liquid dressing, comprising: mixing and reacting naringin and copper salt, centrifuging, discarding the supernatant, adding tannic acid, and ultrasonically stirring to obtain the dressing. This invention first constructs a stable naringin-copper ion nanonetwork structure through intermolecular forces. Based on this, it further utilizes the complexation reaction between tannic acid and copper ions for hybridization, which not only improves the stability of the material but also endows it with the ability to actively capture bacteria in the wound environment and achieve stable adhesion to the wound surface. Furthermore, tannic acid hybridization enables the sustained release of copper ions, thereby effectively improving the biocompatibility of the material and providing a better option for wound repair.

[0025] The innovative aspects of this invention, "tannin-hybridized naringin-copper ion nanonetwork"

[0026] (1) Unique three-dimensional adhesion network structure design

[0027] Naringin and copper ions form an ordered three-dimensional network structure through self-assembly. Tannic acid hybridization further enhances the material's structural stability and biocompatibility, creating a multi-layered adhesive network. This network structure, through the steric hindrance and charge stabilization effects of hybridization, effectively inhibits particle aggregation, ensuring that nanoparticles fully exhibit their nanoscale effects and enhancing the overall performance of the material.

[0028] (2) Synergistic antibacterial and healing-promoting effects through multiple mechanisms

[0029] Copper ions trigger the Fenton reaction, generating highly oxidizing hydroxyl radicals (·OH), which directly kill bacteria by damaging their DNA and cell membranes. Naringin assists in antibacterial activity by inhibiting bacterial biofilm formation and enhancing copper ion permeability; tannins further enhance the antibacterial effect by disrupting bacterial cell walls through molecular complexation. Naringin also possesses anti-inflammatory properties, reducing wound inflammation and synergistically working with copper ions to accelerate angiogenesis, promote collagen deposition, and expedite wound repair and regeneration. This multi-mechanism synergistic antibacterial and healing-promoting effect is more effective than single antibacterial agents, targeting a wider range of bacterial species, including some drug-resistant bacteria.

[0030] (3) Optimization of biocompatibility and stability

[0031] Tannic acid stabilizes the nanonetwork through non-covalent interactions (such as hydrogen bonds and hydrophobic interactions), effectively preventing copper ion leakage. Copper ions play an important role in wound healing, such as antibacterial activity and promoting angiogenesis; however, excessively high local concentrations can cause toxicity. By regulating the network structure, copper ions can be released slowly at an appropriate rate, sustaining their biological activity to meet the needs of wound healing while avoiding the toxic side effects on surrounding healthy tissues caused by excessively high local concentrations due to instantaneous release, thus effectively improving the biosafety of the material.

[0032] (4) Potential for clinical application

[0033] On the one hand, it helps reduce antibiotic use, thereby reducing the risk of drug resistance; at the same time, due to the use of natural products, the cost is lower, which can effectively reduce the overall treatment cost; on the other hand, it has excellent stability, which can ensure that its performance remains stable during storage and use; and its good adhesion can enhance its actual effect in complex wound environments, especially suitable for chronic or difficult-to-heal wounds such as diabetic foot ulcers, infections, and burns.

[0034] The purpose of this invention is to integrate natural active ingredients with nanotechnology to construct a multi-mechanism synergistic and biocompatible infectious wound treatment system, thereby addressing the core pain points of existing technologies.

[0035] (1) Provide non-antibiotic-dependent antimicrobial regimens for infections caused by drug-resistant bacteria (such as MRSA and Pseudomonas aeruginosa).

[0036] Copper ions generate hydroxyl radicals (·OH) through the Fenton reaction, directly damaging bacterial DNA and cell membranes. Naringin, on the other hand, inhibits biofilm formation, blocking bacterial escape routes, thus forming a dual "physical-chemical" killing mechanism. This avoids antibiotic resistance caused by overuse, and copper ions have a broad antibacterial spectrum, effective against both Gram-positive and Gram-negative bacteria.

[0037] (2) Reduce the toxicity of metal ions

[0038] This study addresses the cytotoxicity issues caused by burst release of traditional copper-based materials (such as copper nanoparticles). Tannic acid, as a natural chelating agent, controls the release rate of copper ions through dynamic complexation, triggering disintegration only under the conditions of an infection microenvironment (acidic + high ROS), thus achieving "on-demand release." After modification with tannic acid, the cytotoxicity of the naringin-copper ion nanonetwork is significantly reduced.

[0039] (4) Promotes rapid healing and functional recovery of infected wounds.

[0040] This addresses the problem that traditional materials only focus on antibacterial properties while neglecting tissue repair.

[0041] Multi-target healing mechanism: Tannins, in synergy with naringin, scavenge ROS and reduce oxidative stress damage to fibroblasts. Naringin is metabolized into naringenin, which reduces the release of inflammatory factors, regulates the body's inflammatory response, and creates a favorable internal environment for subsequent tissue repair. Copper ions: As a cofactor of angiogenic factors (such as VEGF), they work with naringenin to promote angiogenesis and improve local microcirculation.

[0042] (5) Improve the biocompatibility and clinical safety of materials

[0043] The design is primarily based on natural ingredients, avoiding potential immune responses to synthetic materials (such as polylactic-co-glycolic acid copolymer, PLGA). It addresses the risk of long-term retention of metal nanoparticles in the body; tannins can be enzymatically broken down into gallic acid and glucose, naringin metabolites are coumaric acid and glucuronic acid, and copper ions are excreted through urine.

[0044] (6) Integrated dressing design

[0045] Integrating antibacterial, anti-inflammatory, and tissue repair functions into a single nanonetwork hybrid, it can directly cover wounds without the need for frequent replacement. Attached Figure Description

[0046] Figure 1 The results show the determination of particle size, potential, polydispersity index, copper and naringin assembly rate of materials with different molar ratios in Example 2;

[0047] Figure 2 The figures show the characterization and performance comparison of the nanonetwork structure of Example 1 and Comparative Examples 1 and 2 of this invention.

[0048] Figure 3 This is a comparison diagram of the surface morphology and roughness of the nano-network in Example 1 and Comparative Example 1 of the present invention;

[0049] Figure 4 This is a Fourier Transmission Infrared (FTIR) spectrum.

[0050] Figure 5 XPS spectra of Example 1 and Comparative Example 1;

[0051] Figure 6 This is a schematic diagram of the molecular docking between tannic acid and the penicillin-binding protein PBP2a of methicillin-resistant Staphylococcus aureus (MRSA) in this invention, and a comparison diagram of the antibacterial effects of the nanocomposite.

[0052] Figure 7 This is a graph showing the results of antibacterial activity;

[0053] Figure 8 The graph shows the results of antioxidant activity.

[0054] Figure 9This is a graph showing the results of anti-inflammatory activity.

[0055] Figure 10 An image showing the effect of angiogenesis in HUVECs cells;

[0056] Figure 11 This is a diagram of a biocompatibility experiment;

[0057] Figure 12 This is a diagram showing the results of an animal experiment. Detailed Implementation

[0058] This invention provides a naringin-copper ion nanonetwork liquid dressing, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The method and application of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the method and application described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0059] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0060] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0061] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0062] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0063] This invention provides a method for preparing a naringin-copper ion nanonetwork liquid dressing, comprising:

[0064] The mixture of naringin and copper salt is centrifuged, the supernatant is discarded, tannic acid is added, and the mixture is ultrasonically stirred to obtain the final product.

[0065] Step 1: Preparation of naringin solution

[0066] First, naringin is prepared into a naringin solution; the naringin solution is a sodium hydroxide solution of naringin. Specifically, naringin is dissolved in a sodium hydroxide solution, which provides an alkaline environment that facilitates the dissolution of naringin and its subsequent reaction with copper ions. Preferably, the concentration of the sodium hydroxide solution is 50 mM. In one embodiment, 23.2 mg of naringin is dissolved in 10 mL of 50 mM sodium hydroxide solution to obtain a 4 mM naringin solution. After dissolving, the naringin is sonicated for 10 minutes, cooled, and then used or left overnight for use the next day.

[0067] Step 2: Mixed reaction of naringin and copper salt

[0068] The naringin solution prepared in step one is mixed with a copper salt solution and reacted. Naringin molecules contain multiple active functional groups such as hydroxyl groups, which can undergo coordination reactions with copper ions to form a preliminary complex. Water-soluble copper salts can be selected, such as common copper salts like copper sulfate, copper chloride, and copper nitrate. During the reaction, the functional groups of naringin interact with copper ions through coordination bonds, gradually forming aggregates with a specific structure.

[0069] In some embodiments, the molar ratio of the naringin and copper salt is 2:1 to 1:8. Specifically, it can be 2:1, 1:1, 1:2, 1:4, or 1:8.

[0070] The complexation ratio of naringin to copper ions needs to be precisely controlled to avoid the side effect of copper ions promoting lipid peroxidation. Molar ratios of naringin to copper salts of 2:1, 1:1, 1:2, and 1:4 show low assembly rates and no significant Tyndall effect.

[0071] The concentration of copper ions in the copper salt is 2-3 mg / mL;

[0072] In some embodiments, one embodiment, the concentration of copper ions is 2.032 mg / mL;

[0073] In some embodiments, the copper salt includes copper sulfate, copper nitrate, or copper chloride.

[0074] In some embodiments, the reaction of naringin and copper salt is an ultrasonic reaction, and the ultrasonication time is 3-10 minutes; specifically, it can be...

[0075] The reaction time can be 3 min, 5 min, 8 min, or 10 min; the ultrasonic power can be set to 200~400W, such as 250W, 300W, or 350W, to ensure sufficient contact between naringin and copper ions and promote the coordination reaction. The reaction temperature can be controlled at room temperature (25±2℃), without the need for additional heating or cooling, making the operation simple.

[0076] During the reaction, the solution color can be observed to gradually change from the initial light yellow to yellowish-brown and become more transparent. This is due to the formation of a coordination complex between naringin and copper ions. The reaction progress can be judged by monitoring the changes in characteristic absorption peaks with a UV-Vis spectrophotometer. Usually, a new absorption peak will appear in the range of 400~600nm, and the peak intensity will gradually increase with the extension of reaction time until a stable state is reached.

[0077] Step 3: Centrifugation

[0078] Centrifuge the mixture from step two. Centrifugation separates the initial complex particles from the unreacted solution components. The centrifugation speed and time can be adjusted according to the actual reaction system to ensure effective separation. After centrifugation, discard the supernatant and retain the precipitate at the bottom, which is the initial complex formed by the reaction of naringin and copper ions.

[0079] The preferred centrifugation speed is 12000-14000 rpm, and the centrifugation time is 20-30 min; specifically, it can be 12000 rpm, 13000 rpm or 14000 rpm, and the centrifugation time can be 20 min, 25 min or 30 min.

[0080] Preferably, the centrifugation speed is 14,000 rpm for 20 minutes. For example, setting the centrifugation speed to 14,000 rpm and the centrifugation time to 20 minutes can effectively settle the smaller initial complex particles while avoiding particle structure damage due to excessive centrifugation intensity. During centrifugation, 1.5 mL centrifuge tubes can be used to evenly distribute the reaction mixture, ensuring balanced forces during centrifugation. After centrifugation, the supernatant is usually light yellow-green. At this time, it should be carefully poured out or pipetteed to remove the supernatant, avoiding stirring the bottom precipitate.

[0081] Step 4: Add tannic acid and mix with ultrasonic stirring;

[0082] Tannic acid is added to the precipitate obtained in the previous step. Tannic acid molecules contain numerous phenolic hydroxyl groups, exhibiting strong complexing and reducing abilities. These hydroxyl groups can further interact with copper ions and naringin molecules in the precipitate, regulating the formation of the nanonetwork structure. After adding tannic acid, the mixture is ultrasonically stirred. Ultrasonic treatment provides sufficient energy to promote thorough mixing and reaction between tannic acid and the precipitate, contributing to the formation of a uniform and stable nanonetwork liquid dressing.

[0083] The uniformity of tannic acid-copper ion hybridization directly affects its performance. Weak intermolecular interactions (hydrogen bonds, coordination bonds) can be used to construct nanonetworks of controllable size, ensuring uniform hybridization coverage. Stirring speed and time are critical control points; excessive stirring speed or time will cause the metal polyphenol network structure to disintegrate.

[0084] In some embodiments, the concentration of the tannic acid is 3-5 mg / mL, the pH value is 7.2, and the amount added is 1 mL.

[0085] After addition, place the mixture in an ultrasonic cleaner for ultrasonic stirring. Set the ultrasonic power to 200W and the ultrasonic time to 10-13 minutes. During this time, remove the mixture and manually help it mix to ensure that the tannic acid and the initial complex are in full contact.

[0086] Add tannic acid to adjust the pH to weakly alkaline (pH=7.2), sonicate for 10 min, stir at 400 rpm for 2-4 h; the centrifugation speed is preferably 12000~14000 rpm for 20~30 min; specifically, it can be 12000 rpm, 13000 rpm or 14000 rpm, and the centrifugation time can be 20 min, 25 min or 30 min.

[0087] After centrifugation, the sample was resuspended in ddH2O.

[0088] This invention provides a naringin-copper ion nanonetwork liquid dressing, which is prepared by any one of the preparation methods described in the above technical solutions.

[0089] In this liquid dressing, naringin molecules interact with copper ions (Cu) through multiple hydroxyl groups in their structure. 2＋ Coordination occurs, forming stable five- or six-membered chelate rings, which then self-assemble into nanoparticles with a three-dimensional network structure. Their microstructure typically presents as irregular sheet-like or spherical aggregates with a narrow particle size distribution. This unique nanonetwork structure endows the material with a large specific surface area, exposing more active sites, thus exhibiting excellent performance in fields such as catalysis, adsorption, and biomedicine.

[0090] This invention provides the application of the naringin-copper ion nanonetwork liquid dressing described above in the preparation of products that promote wound healing and / or improve healing quality.

[0091] The wound-healing-promoting properties include one or more of the following: anti-inflammatory, antioxidant, and antibacterial.

[0092] The improvement in healing quality includes one or more of the following: promoting granulation tissue growth, accelerating collagen deposition, reducing scar formation, and improving skin barrier function. The improvement in healing quality also includes promoting tissue regeneration and angiogenesis.

[0093] This invention provides a product that promotes wound healing and / or improves the quality of healing, including the naringin-copper ion nanonetwork liquid dressing described in the above technical solution.

[0094] The product described in this invention can be a drug, medical device, skincare product, or health product. In the pharmaceutical field, the liquid dressing can be prepared into dosage forms such as a topical gel, cream, spray, or dressing, and applied topically to the wound to directly promote healing. In the medical device field, it can be loaded onto carriers such as medical sponges, gauze, or biomembranes to create wound repair materials with active repair functions. In the skincare product field, it can be added to products such as creams and serums with repairing effects to improve issues such as micro-wounds and sensitive skin repair. In the health product field, it can be used as a functional ingredient to assist in promoting the repair and regeneration of body tissues through oral or topical application.

[0095] This invention first forms a nano-network structure by complexing naringin and copper ions, followed by tannic acid hybridization. This enhances the material's stability and biocompatibility, and endows it with the ability to capture bacteria. The three components synergistically achieve an integrated antibacterial, anti-inflammatory, and wound-healing function, playing a simultaneous role in the healing of infected wounds and accelerating wound healing.

[0096] This invention constructs a stable naringin-copper ion nanonetwork structure through intermolecular forces (such as hydrogen bonds and complexation), and further utilizes the complexation reaction between tannic acid and copper ions to hybridize it, thereby enhancing its hydrophilicity and facilitating the wettability and cell adhesion of the material in wound environments. The stable hybridization can inhibit the aggregation of nanoparticles and achieve the sustained release of copper ions, avoiding toxicity caused by excessively high local concentrations.

[0097] The present invention has the following beneficial effects:

[0098] (1) Antibacterial and biofilm removal:

[0099] The phenolic hydroxyl groups in tannins can bind to bacterial cell membranes, enabling them to better capture bacteria. Naringin can disrupt the bacterial cell membrane structure, leading to leakage of cell contents and bacterial death. Copper ions can also disrupt the bacterial cell wall and membrane structure, causing leakage of cell contents and bacterial death. Furthermore, they can generate reactive oxygen species through redox reactions, damaging bacterial macromolecules such as DNA, RNA, and proteins, leading to bacterial death. In addition, they can enter the bacterial cell interior, inhibiting enzyme activity and interfering with bacterial metabolic processes, thereby inhibiting their growth and reproduction. These three substances form a dynamic complex structure, doubly inhibiting biofilm formation and disintegrating mature colonies by disrupting bacterial cell membrane permeability and reducing bacterial adhesion to host cells.

[0100] (2) Regulation of oxidative stress: Naringin-copper ion nanonetwork has natural oxidase activity, which catalyzes the decomposition of excess reactive oxygen species (ROS), reduces the level of intracellular oxidative stress, and protects cells from damage;

[0101] Tannins scavenge reactive oxygen species (ROS), reducing oxidative stress damage to fibroblasts and keratinocytes; naringin regulates the expression of antioxidant defense proteins, increasing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX) in vivo. Antioxidant activity protects normal cells around wounds, reducing the adverse effects of oxidative damage on the healing process.

[0102] (3) Inflammation-repair coupling: Naringin inhibits the release of inflammatory factors, while synergistically promoting angiogenesis and accelerating collagen deposition with copper ions; naringin downregulates the expression of pro-inflammatory factors (such as TNF-α and IL-6) and upregulates the expression of anti-inflammatory factors (such as IL-10), accelerating the transition from the inflammatory phase to the proliferative phase; tannins have anti-inflammatory properties, reducing the interference of the inflammatory response on wound healing. The multi-mechanism anti-inflammatory effect can more comprehensively control the inflammatory response and promote wound healing.

[0103] This invention combines naringin, copper ions, and tannic acid to form a liquid dressing, which can fully utilize the bioactivity of the three to achieve a multifunctional synergistic effect. It can be applied to the treatment of infected wounds, and accelerates the recovery process by inhibiting bacterial infection, reducing inflammatory response, and promoting wound healing.

[0104] Synergistic antibacterial action: Copper ions generate hydroxyl radicals (·OH) through the Fenton reaction, damaging bacterial DNA and cell membranes, leading to bacterial death. Naringin directly inhibits the proliferation of pathogens, interfering with their acid production, sugar production, and adhesion functions, thus weakening their survival ability. Tannic acid binds to the bacterial cell wall (peptidoglycan) and membrane phospholipids through hydrogen bonds and π-π stacking, disrupting membrane integrity and inducing leakage of intracellular contents. Naringin disrupts the bacterial growth environment, copper ions directly kill bacteria through oxidative stress, and tannic acid damages the bacterial cell wall; these three elements form a three-dimensional antibacterial network of "environmental interference-oxidative damage-physical destruction." Furthermore, tannic acid and copper ions inhibit bacterial biofilm formation, and naringin enhances the efficacy of antibiotics, collectively reducing the risk of bacterial resistance.

[0105] Synergistic anti-inflammatory effects: Naringin downregulates the expression of pro-inflammatory factors such as IL-6, IL-1β, and TNF-α, blocks the NF-κB and NLRP3 inflammatory signaling pathways, and alleviates the inflammatory response. Copper, as a cofactor of superoxide dismutase (SOD), participates in the scavenging of ROS and reduces oxidative stress-induced inflammatory damage. Copper ions can promote the polarization of M2 macrophages, shifting them from a pro-inflammatory state (M1) to an anti-inflammatory and pro-repair state (M2), accelerating inflammation resolution. Tannins scavenge ROS and protect cells from oxidative damage. These agents inhibit inflammation from multiple dimensions: signal regulation, immune regulation, and oxidative stress.

[0106] Synergistic healing effects: Naringin upregulates VEGF expression, activates the PI3K / Akt signaling pathway, promotes angiogenesis, and provides sufficient nutrition to the wound. Copper ions stimulate endothelial cell proliferation and migration, promote vascular network formation, and improve local blood supply to the wound. Tannic acid-functionalized materials enhance keratinocyte migration and accelerate epidermal regeneration. These three components form a complete repair chain of "migration promotion-vascularization-tissue regeneration," and can regulate collagen deposition. Naringin inhibits excessive fibrosis, and copper ions promote orderly angiogenesis, collectively reducing the risk of scarring.

[0107] Functional stability: Tannin hybridization prevents the aggregation and oxidation of naringin and copper nanonetworks, ensuring the stability of the nanonetworks; in addition, it enables the nanonetworks to capture bacteria more efficiently, improving the stability of antibacterial function.

[0108] This invention achieves highly efficient antibacterial, antioxidant, anti-inflammatory, and tissue regeneration-promoting effects through the synergistic effect of multiple mechanisms.

[0109] Synergistic antibacterial action through multiple mechanisms:

[0110] Copper ions (Cu) 2＋ ): It generates hydroxyl radicals (·OH) through the Fenton reaction, which directly damage bacterial DNA and cell membranes, while inhibiting biofilm formation. It is effective against drug-resistant bacteria (such as MRSA and Pseudomonas aeruginosa).

[0111] Tannic acid (TA): As a natural antibacterial agent, it destroys bacterial cell walls through molecular complexation, enhances the antibacterial spectrum, and reduces cytotoxicity caused by the burst release of copper ions.

[0112] Naringin: It inhibits some bacteria such as Pseudomonas spp., and at the same time reduces local inflammation in wounds and lowers the risk of infection through its anti-inflammatory effect.

[0113] Healing-promoting properties:

[0114] (1) Antioxidant and anti-inflammatory effects: Tannins and naringin scavenge ROS, reduce oxidative stress damage to cells, and promote wound healing. Naringin downregulates pro-inflammatory factors (such as TNF-α and IL-6) and upregulates anti-inflammatory factors (such as IL-10), accelerating the transition from the inflammatory phase to the proliferative phase.

[0115] (2) Promotes angiogenesis and tissue regeneration: Naringin and a small amount of copper ions accelerate the formation of new blood vessels and stimulate collagen deposition.

[0116] Biocompatibility and biodegradability: Tannins and naringin are both natural components, and their degradation products (such as gallic acid and glucuronic acid) are non-toxic and can be excreted in urine. Copper ion release is controlled within a safe range to avoid heavy metal accumulation.

[0117] This invention utilizes a nanonetwork structure constructed through the dynamic complexation of tannic acid-hybridized naringin and copper ions, ensuring its stability. It remains stable in normal tissues, triggering disintegration only at the infection site. The on-demand release mechanism of copper ions in the infection microenvironment avoids the cytotoxicity caused by burst release in traditional copper-based materials. Furthermore, the antioxidant effects of tannic acid and naringin are combined to reduce damage to normal cells from high ROS. Tannic acid and naringin chelate copper ions, jointly exerting antibacterial activity and reducing damage to cell-forming cells from free copper ions. The structural stability of naringin in the nanonetwork ensures that its anti-inflammatory and angiogenic activities are not affected by the preparation process, improving the bioavailability of naringin and prolonging its duration of action.

[0118] Tannic acid and naringin are both natural components, and their degradation products (such as gallic acid and glucuronic acid) are non-toxic and can be excreted in urine. Using natural components reduces the potential side effects and toxicity of chemically synthesized drugs. The release of copper ions is controlled within a safe range to avoid heavy metal accumulation. By rationally regulating the dosage and release rate of copper ions, antibacterial effects can be achieved while reducing toxicity to normal cells.

[0119] Nanonetwork structure design: A nanonetwork structure is constructed through the dynamic complexation of tannic acid and copper ions to ensure its stability and intelligent response performance. The optimized nanonetwork structure can improve the adhesion and stability of the material, making it better suited for application on wound surfaces.

[0120] Process parameter control: This invention precisely controls the preparation process parameters. Precise process parameter control can improve the quality and consistency of the product and ensure the stability of the therapeutic effect.

[0121] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0122] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0123] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0124] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0125] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0126] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides a naringin-copper ion nanonetwork liquid dressing, its preparation method, and its application.

[0127] Instruments and detection methods used in all embodiments of the present invention

[0128] Naringin was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); anhydrous copper sulfate was purchased from Tianjin Kemei Chemical Reagent Co., Ltd. (Tianjin, China); tannic acid was purchased from Shanghai Shanpu Chemical Co., Ltd. (Shanghai, China); bacterial live / dead staining kit and hematoxylin-eosin (H&E) staining kit were purchased from Beyotime Biotechnology Co., Ltd. (Shanghai, China); sodium hydroxide was purchased from Hunan Huihong Reagent Co., Ltd. (Changsha, China); matrix gel was purchased from Yisheng Biotechnology (Shanghai) Co., Ltd. (Shanghai, China); interleukin-10 (IL-10) was purchased from [unclear - likely a specific reagent or reagent]. 10) The enzyme-linked immunosorbent assay (ELISA) kit for tumor necrosis factor-α (TNF-α) was purchased from Shenzhen Xinbosheng Biotechnology Co., Ltd. (Shenzhen, China); lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (Shanghai, China); DCFH-DA reactive oxygen species probe and thiazolyl blue (MTT) were purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China); dimethyl sulfoxide (DMSO) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China); phosphate-buffered saline (PBS) was purchased from Wuhan Pronosei Life Science Co., Ltd. (Wuhan, China); methicillin-resistant Staphylococcus aureus (MRSA) was obtained from Hunan Provincial Center for Disease Control and Prevention; and human umbilical vein endothelial cells (HUVECs) and mouse mononuclear macrophage leukemia cells (RAW264.7) were obtained from the cell bank of Xiangya Hospital, Central South University (Changsha, China).

[0129] All experiments used double-deionized water (ddH2O). BALB / c and DB / DB mice were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. Animal experiments were conducted in accordance with the ethical guidelines and protocols approved by the Medical Ethics Committee of Hunan University (SYXK-2023-0010).

[0130] The zeta potential and particle size distribution of nanoparticles were measured using a dynamic light scattering (DLS, Malvern Instruments, Zetasizer Nano ZSP). The assembly amount of naringin (NRG) was determined at 282 nm using a UV-Vis spectrophotometer (Shimadzu, UV2700). The assembly quality of copper ions was determined using ICP-OES (Thermo ICP-OES 7200, Thermo Fisher Scientific). The morphology of the nanomaterials was characterized using low-pressure transmission electron microscopy (TEM, FEI Tecnai G2 spirit, FEI Corporation). The surface roughness of the material was characterized using atomic force microscopy (AFM, Burker Dimension FastScan / Icon, Bruker (Beijing) Technology Co., Ltd.). The absorption peak changes were measured using FTIR spectroscopy (Thermo Fisher Scientific, Nicolet 6700 FT-IR spectrometer). The valence state of copper ions in the material was determined using X-ray photoelectron spectroscopy (Thermo Fisher Scientific, Thermo escalab). 250Xi), absorbance was measured using a microplate reader (Eprie, Platinum Elmer Instruments Ltd.), and cell and section morphology were observed using an inverted microscope (Olympus Corporation, IX-73).

[0131] The concentrations of the individual naringin solutions in all figures of this invention are: 4 mmol / L NRG (solvent pH=12.7, c(NRG)=2.32 mg / mL); the concentrations of the individual copper sulfate solutions are: 32 mmol / L CuSO4 solution (c(Cu)=2.32 mg / mL). 2＋ (2.032 mg / mL)

[0132] Example 1:

[0133] Synthesis of tannic acid-hybridized copper-naringin nanonetwork: 0.5 mL of 4 mmol / L NRG solution (solvent pH=12.7, c(NRG)=2.32 mg / mL) was added to 0.5 mL of 32 mmol / L CuSO4 solution (c(Cu)=2.32 mg / mL). 2+ Mix the solution with 2.032 mg / mL of TA (pH=2.032), sonicate for 5 min to mix thoroughly, centrifuge at 14000 rpm for 20 min, discard the supernatant, add 1 mL of 4 mg / mL TA (pH=7.2) to adjust the pH, and sonicate for 10 min to mix thoroughly. Take 2 mL of the mixture, stir at 400 rpm for 4 h, centrifuge at 14000 rpm for 30 min, discard the supernatant, and resuspend in ddH2O.

[0134] Example 2:

[0135] Synthesis of copper-naringin nanonetwork: 0.5 mL of 4 mmol / L naringin (NRG) (solvent pH=12.7, c(NRG)=2.32 mg / mL) was added to 0.5 mL of 2, 4, 8, 16, and 32 mmol / L CuSO4 (c(Cu) = 2.32 mg / mL). 2+ In a solution containing 2.032 mg / mL naringin, the mixture was sonicated for 5 min to mix thoroughly, then centrifuged at 14000 rpm for 20 min. The supernatant was discarded, and the solution was resuspended in ddH2O. The molar ratios of naringin and copper were 2:1, 1:1, 1:2, 1:4, and 1:8, respectively.

[0136] Figure 1 The results of particle size, potential, polydispersity index, and assembly rates of copper and naringin in materials with different molar ratios in Example 2 are shown, along with corresponding images of the actual materials. Where a represents the particle size of NRG-Cu MPNs with different molar ratios, b represents the zeta potential of NRG-Cu MPNs with different molar ratios, c represents the polydispersity index (PDI) of NRG-Cu MPNs with different molar ratios, d represents the assembly rate of copper in NRG-Cu MPNs with different molar ratios, e represents the assembly rate of naringin in NRG-Cu MPNs with different molar ratios, and f represents the Tyndall effect of the mixture of naringin and copper sulfate. Finally, a 1:8 ratio was selected as the optimal ratio, resulting in the highest assembly rate of naringin and copper ions.

[0137] Comparative Example 1:

[0138] Synthesis of copper-naringin nanonetwork: 0.5 mL of 4 mmol / L naringin (NRG) (solvent pH=12.7, c(NRG)=2.32 mg / mL) was added to 0.5 mL of 32 mmol / L CuSO4 (c(Cu) = 2.32 mg / mL). 2+ In a solution with a concentration of 2.032 mg / mL, the mixture was sonicated for 5 min to mix thoroughly, then centrifuged at 14000 rpm for 20 min. The supernatant was discarded and the solution was resuspended in ddH2O.

[0139] Comparative Example 2:

[0140] Tannic acid hybrid copper-naringin at different stirring times or speeds: 0.5 mL of 4 mmol / L NRG solution (solvent pH=12.7, c(NRG)=2.32 mg / mL) was added to 0.5 mL of 32 mmol / L CuSO4 solution (c(Cu)=2.32 mg / mL). 2+In a solution containing 2.032 mg / mL TA (pH=2.032), sonicate for 5 min to mix thoroughly, then centrifuge at 14000 rpm for 20 min, discard the supernatant, add 1 mL of 4 mg / mL TA (pH=7.2) to adjust the pH, and sonicate for 10 min to mix thoroughly. Take 2 mL of the mixture, stir at 400 rpm / 600 rpm for 6 h, then centrifuge at 14000 rpm for 30 min, discard the supernatant, and resuspend in ddH2O.

[0141] Figure 2 In Figure 1, a represents the copper-naringin nanonetwork of Comparative Example 1, b represents the copper-naringin nanonetwork hybridized with tannins in Example 1, c represents the results of Comparative Example 2, a, b, and c are transmission electron microscopy (TEM) images, and in d and e, blue represents Comparative Example 1 and red represents Example 1. d represents the particle size of both, and e represents the Zeta potential of both. TEM observations of a and b, i.e., Comparative Example 1 and Example 1, revealed that the nanonetwork structure remained unchanged before and after tannin hybridization. The average particle size after hybridization in d was essentially the same as before hybridization, indicating that tannin hybridization did not destroy the nanostructure of naringin-copper ions. Observation in c showed that the hybridized nanonetwork structure disintegrated when the stirring speed or stirring time was too high in Comparative Example 2. Potential detection showed that the potential of the hybridized nanocomposite changed from -11.68±0.43 to -36.3±0.89, indicating that tannin hybridization allows the nanocomposite to be more uniformly dispersed in solution, reducing precipitation and aggregation.

[0142] Figure 3 In Figure a1, the copper-naringin nanonetwork of Comparative Example 1 is shown, and in Figure a2, the copper-naringin nanonetwork hybridized with tannins is shown in Example 1. Figure a is an atomic force microscopy (AFM) image. In Figures b and c, blue represents Comparative Example 1, and red represents Example 1. Figures b and c show the quantitative analysis of the AFM results. AFM analysis of the surface morphology of the samples before and after hybridization revealed that the nanocomposite network structure remained unchanged. After hybridization, the surface roughness of the samples significantly increased, with Ra and Rq increasing from 0.81 ± 0.51 nm and 1.039 ± 0.56 nm in Comparative Example 1 to 3.69 ± 2.72 nm and 5.51 ± 3.34 nm in Example 1, respectively. This indicates that tannins were successfully modified into the copper-naringin nanonetwork via ligand exchange.

[0143] Infrared spectroscopy measurements were performed on the products of Example 1 and Comparative Example 1, and the results are as follows: Figure 4 The above, Figure 4 The image shows the Fourier Transform Infrared (FTIR) spectrum, where black represents naringin, red represents the copper-naringin nanonetwork of Comparative Example 1, and blue represents the tannic acid-hybridized copper-naringin nanonetwork of Example 1. The infrared spectrum of naringin at 1645 cm⁻¹... -1This is a characteristic absorption peak for the carbonyl group (C=O). After forming a complex with copper (Comparative Example 1), this absorption peak undergoes a blue shift, moving to approximately 1609 cm⁻¹. -1 This indicates that the carbonyl oxygen atom in the naringin molecule forms a coordinate bond with the copper ion, altering the vibrational frequency of the carbonyl group. After the complexation reaction, 3414 cm⁻¹ -1 The broadening of the absorption peak at 1600-1500 cm⁻¹ reflects the interaction between the hydroxyl group and the copper ion; -1 The regional absorption peaks did not change significantly before and after coordination, indicating that the benzene ring structure was not destroyed. After tannic acid hybridization (Example 1), the surface of the naringin-copper nanonetwork showed an absorption peak at 1707 cm⁻¹. -1 A new absorption peak appears at 2922 cm⁻¹, corresponding to the C=O bond of the carboxyl or carbonyl group, proving the existence of hybridization; simultaneously, 2922 cm⁻¹ -1 The disappearance of the absorption peak indicates that tannic acid (TA) interacts with naringin-copper (NRG-Cu), altering the original CH vibration mode.

[0144] XPS spectroscopy measurements were performed on the products of Example 1 and Comparative Example 1, and the results are as follows: Figure 5 The above, Figure 5 The images are XPS spectra, where a represents Comparative Example 1 and b represents Example 1. Analysis of the X-ray photoelectron spectroscopy revealed that copper ions in both Comparative Example 1(a) and Example 1(b) were predominantly divalent.

[0145] Figure 6 Figure a shows a schematic diagram of molecular docking between tannic acid and PBP2a, the penicillin-binding protein of methicillin-resistant Staphylococcus aureus (MRSA). Figure b shows a photograph on the left of Comparative Example 1, co-cultured with MRSA for 12 hours, and on the right of Example 1, co-cultured with MRSA for 12 hours. PBP2a (penicillin-binding protein PBP2a) is a key protein in MRSA resistance. Molecular docking results show that tannic acid occupies the active sites GLN43, ALA46, and ASP47 of PBP2a through hydrogen bonding, hydrophobic interactions, or π-π stacking, with a docking score of -6.488 kcal / mol, demonstrating that tannic acid has the function of capturing bacteria. The co-culture experiment with bacteria (Figure b) shows that hybridization makes it easier to adsorb bacteria.

[0146] Figure 7 To determine the antibacterial activity results, the experiment used 1*10 8 MRSA bacterial suspensions at CFU / mL were co-cultured with different materials for the same time. The mixtures of MRSA and materials were then diluted and plated to determine their antibacterial properties. Results are as follows: Figure 7 The above, Figure 7Image a shows a diluted plate photograph of the antibacterial activity experiment, and image b shows the antibacterial rate statistics. The results show that at the same concentration, NRG has virtually no antibacterial activity, while CuSO4 and NRG-Cu MPNs (Comparative Example 1) have certain antibacterial activity, with antibacterial rates of 59.6% and 72.7%, respectively. After tannic acid hybridization, NRG-Cu MPNs@TA (Example 1) further enhanced its antibacterial activity, with an antibacterial rate of 93.5%.

[0147] Figure 8 To determine the antioxidant activity results, the experiment included a control group (normal cells without LPS or the treatment material), a model group (cells treated with only 100 ng / mL LPS), and other intervention groups (NRG, CuSO4, TA, NRG-Cu MPNs (Comparative Example 1), and NRG-Cu MPNs@TA (Example 1)) which were cells pretreated with the treatment material for 2 hours and then co-incubated with LPS. All groups were incubated with reactive oxygen species (ROS) probes for 30 minutes, and the cells were imaged using a live-cell fluorescence imaging system (LPS induces the production of ROS in cells). The antioxidant activity results are as follows: Figure 8 In the figures, a represents the fluorescence imaging and bright-field image of ROS-cleared RAW264.7 cells, and b represents the quantitative analysis of fluorescence intensity. ControlL cells were normal cells without LPS induction, while Model cells were cells subjected to induced oxidative stress without any material treatment. Compared to the model group, the hybridized material (Example 1) significantly regulated intracellular reactive oxygen species (ROS) levels, substantially reducing ROS content. The reduced ROS levels were not significantly different from the control group (normal cells in the ControlL group), meaning they recovered to levels close to the control group.

[0148] Figure 9 To determine the anti-inflammatory activity, the experiment included a control group (normal cells without LPS or the treatment material), a model group (cells treated with 100 ng / mL LPS for 24 h), and other intervention groups (NRG and NRG-CuMPNs@TA (Example 1) cells pretreated with the treatment material for 2 h followed by LPS co-incubation (LPS can induce cellular oxidative stress, triggering an inflammatory cascade). The levels of inflammatory factors in the supernatant of LPS-induced RAW264.7 cells were measured using an enzyme-linked immunosorbent assay (ELISA) kit for interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α). Control was the supernatant of normal cells without LPS induction, and Model was the supernatant of cells with LPS-induced inflammation. Results are as follows: Figure 9 As shown: Figure 9In the figure, a represents the TNF-α level in RAW264.7 cells, and b represents the IL-10 level in RAW264.7 cells. The results showed that compared with the model group, the hybridized material (Example 1) could significantly regulate the anti-inflammatory activity of cells, significantly decreasing the level of the pro-inflammatory factor TNF-α and significantly upregulating the level of the anti-inflammatory factor IL-10.

[0149] Figure 10 To assess the angiogenesis effect of HUVECs cells, the experiment involved first laying matrix gel at the bottom of well plates, followed by cell culture. The experiment included a control group (normal cells without matrix gel) and other intervention groups (NRG, CuSO4, TA, NRG-Cu MPNs (Comparative Example 1), and NRG-Cu MPNs@TA (Example 1)) where matrix gel was added and cells were co-cultured. After 4 hours of culture, the cells were observed under a microscope. Results are as follows: Figure 10 The above, Figure 10 In Figure a, we see a microscopic image of the angiogenesis effect in HUVECs cells; b represents the number of branch nodes; c represents the number of ring structures; and d represents the tube length. Compared with the control group, the hybrid material NRG-Cu@TA MPNs (Example 1) promoted endothelial cell tube formation, and the tube formation level was much higher than that of NRG, CuSO4, and NRG-CuMPNs (Comparative Example 1).

[0150] Figure 11 Biocompatibility tests, including the MTT assay and hemolysis assay:

[0151] In the MTT experiment, the control group consisted of normal cells without any added materials, while the other drug intervention groups (NRG, CuSO4, TA, NRG-Cu MPNs (Comparative Example 1), and NRG-Cu MPNs@TA (Example 1)) were cells incubated with added materials. After 4 hours of treatment, all groups were incubated with MTT for 2 hours, and then MTT was aspirated and DMSO was added. The absorbance at 490 nm was measured using a microplate reader to detect cell viability and proliferation.

[0152] The hemolysis experiment included a positive control group (Water, ultrapure water) and a negative control group (PBS, phosphate buffered saline). Other intervention groups included NRG, CuSO4, NRG-Cu MPNs (Comparative Example 1), and NRG-Cu MPNs@TA (Example 1). The red blood cell suspension was mixed with the test sample of each group and incubated for 4 hours. After centrifugation, the supernatant was collected and the absorbance was measured. The red blood cells were then resuspended in PBS and the cell morphology was observed using an inverted microscope.

[0153] The results are as follows Figure 11In the figures, a and b show the MTT assay results for RAW264.7 cells and HUVECs cells, respectively; c shows the hemolysis assay results and corresponding blood state photographs; and d shows the morphology of erythrocytes in the hemolysis assay. The MTT and hemolysis assay results indicate that the hybrid material (Example 1) has significantly improved safety for macrophages and vascular endothelial cells and did not induce hemolysis, demonstrating its good biocompatibility.

[0154] Figure 12 For animal experiments, a shows images of wounds at different stages, and b shows H&E and Masson staining images of wound sections. Diabetic DB / DB mice were used in the animal experiments and randomly divided into four groups. After establishing an infected wound model, different drugs were administered to each group, with each administration consisting of 100 μL of medication. The Model group received ultrapure water; Vancomycin served as the positive control group at a dose of 2 μg / mL; NRG at a dose of 20 μg / mL; and NRG-Cu@TA MPNs (Example 1) were administered at a dose of 20 μg / mL. Wound morphology changes were observed and recorded periodically (Figure a). At the end of the treatment period, skin tissue sections were collected for H&E and Masson staining (Figure b) to assess wound healing, epithelial regeneration, and collagen deposition. Results showed that compared to the Model group, the hybrid material (Example 1) significantly improved wound healing speed in the animal group. When the wounds of the mice in Example 1 were completely closed, the wound closure rate in the Model group was 58%.

[0155] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing a tannin-hybridized naringin-copper ion nanonetwork liquid dressing, characterized in that, include: Naringin and copper salt are first mixed and reacted, centrifuged, and the supernatant is discarded. Then, tannic acid is added and the mixture is ultrasonically stirred to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The molar ratio of naringin to copper salt is 2:1 to 1:8; The concentration of copper ions in the copper salt is 2~3 mg / mL.

3. The preparation method according to claim 1, characterized in that, The copper salts include copper sulfate, copper nitrate, or copper chloride.

4. The preparation method according to claim 1, characterized in that, The concentration of the tannic acid is 3~5 mg / mL, the pH value is 7.2, and the amount added is 1 mL.

5. The preparation method according to claim 1, characterized in that, The reaction of naringin and copper salt is an ultrasonic reaction, the ultrasonic time is 2-5 min, the power is 150-230 W; the centrifugation is 12000-14000 rpm, and the centrifugation time is 20-30 min.

6. The preparation method according to claim 1, characterized in that, Add tannic acid to adjust the pH to weakly alkaline (pH=7.2), sonicate for 10 min, stir at 400 rpm for 2-4 h; centrifuge at 12000~14000 rpm for 20~30 min; resuspend in ddH2O after centrifugation.

7. A naringin-copper ion nanonetwork liquid dressing, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 6.

8. The use of the naringin-copper ion nanonetwork liquid dressing of claim 7 in the preparation of products that promote chronic wound healing and / or improve healing quality.

9. The application according to claim 8, characterized in that, The wound-healing-promoting properties include one or more of the following: antibacterial, antioxidant, and anti-inflammatory. Improving healing quality includes promoting tissue regeneration and angiogenesis.

10. A product that promotes wound healing and / or improves the quality of healing, characterized in that, Including the naringin-copper ion nanonetwork liquid dressing as described in claim 7.

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