A magnetic particle-protein fiber dressing and a preparation method and application thereof

Abstract

The application belongs to the technical field of scarless skin lesion repair, and particularly relates to a magnetic particle-protein fiber dressing as well as a preparation method and application thereof. The magnetic nanoparticles are coated to obtain coated magnetic nanoparticles, the coated magnetic nanoparticles are mixed with a water-soluble copper salt and water to load copper ions, the coated magnetic nanoparticles loaded with copper ions are mixed with functional proteins and a buffer solution to be incubated, the obtained magnetic microsphere-protein composite solution is placed in an external magnetic field to be magnetically attracted, and then directional assembly is carried out in the external magnetic field to obtain a magnetic particle-protein fiber assembly. The magnetic particle-protein fiber assembly and the medical dressing coated with the magnetic particle-protein fiber provided by the application can realize rapid scarless repair of a wound and restore the integrity of skin function, and the preparation method is simple, and the application has important clinical application value and market prospect.

Description

Technical Field

[0001] This invention belongs to the field of scarless skin damage repair technology, specifically relating to a magnetic particle-protein fiber dressing, its preparation method, and its application. Background Technology

[0002] As the largest and strongest organ in the human body, the skin is the body's first line of defense, capable of resisting external damage or the invasion of pathogenic microorganisms. However, in the face of nature, the skin is also relatively fragile, because it is exposed to the air and can be damaged by factors such as high temperatures, scratches, and biochemical substances. In particular, chronic trauma, burns, and skin wound infections require arduous and long-term treatment, becoming an important cause of seriously endangering people's physical and mental health and reducing their quality of life and survival.

[0003] Clinical wound healing problems are widespread, ranging from "insufficient healing" (i.e., chronic / non-healing wounds) to "over-healing" (i.e., scarring / fibrosis). Wound healing comprises the classic three stages: inflammation, proliferation, and remodeling. New insights suggest that these three stages overlap or occur in parallel with the wound in both time and space (Baron JM, Glatz M, Proksch E. Optimal Support of Wound Healing: New Insights[J]. Dermatology, 2020, 236(6):593-600.). Broadly speaking, dysregulation or interruption at any stage of wound healing can lead to healing exceeding the normal range. Narrowly speaking, they represent the two ends of the wound healing time curve. On one hand, wound healing is slow, and in cases of pressure / tissue ischemia, bacterial inflammation, or immune dysfunction (often caused by systemic factors such as diabetes or atherosclerosis), the wound may not heal at all. Chronic non-healing wounds, on the other hand, remain open to the outside environment, making them highly susceptible to complications, potentially leading to infection from the skin to underlying structures, requiring surgical intervention or even amputation. On the other hand, there are scars formed after excessive healing.

[0004] Currently, implantable medical devices for tissue repair are rapidly developing, with various biomaterials such as antibacterial gauze, cotton balls, hydrogel films, microspheres, and nanofibers being developed. Although these implantable biomaterials generally promote wound healing, scarring caused by the rapid repair of damage remains a pressing problem. This is because implants trigger complex immune responses within the body, simultaneously promoting fibroblast proliferation and excessive collagen deposition, leading to fibrosis. Fibrotic skin is weaker in tensile strength than normal skin and loses its functional integrity.

[0005] In summary, current tissue repair materials cannot yet achieve rapid, scarless skin repair. Summary of the Invention

[0006] The purpose of this invention is to provide a magnetic particle-protein fiber assembly and its preparation method and application, as well as a magnetic particle-protein fiber dressing and its preparation method and application. The magnetic particle-protein fiber assembly and magnetic particle-protein fiber dressing provided by this invention can achieve rapid wound repair without scarring and restore the integrity of skin function; moreover, the preparation method is simple and has important clinical application value and market prospects.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] This invention provides a method for preparing a magnetic particle-protein fiber assembly, comprising the following steps:

[0009] Magnetic nanoparticles are coated to obtain coated magnetic nanoparticles, wherein the coating material of the coated magnetic nanoparticles is a negatively charged polymer.

[0010] The coated magnetic nanoparticles, water-soluble copper salt, and water were mixed to load copper ions, thereby obtaining coated magnetic nanoparticles loaded with copper ions.

[0011] The copper-loaded coated magnetic nanoparticles, functional protein and buffer solution were mixed and incubated to obtain a magnetic microsphere-protein complex solution.

[0012] The magnetic microsphere-protein complex solution was placed in an external magnetic field for magnetic attraction to obtain an intermediate assembly of magnetic composite microspheres.

[0013] The magnetic composite microsphere intermediate assembly was oriented and assembled under the control of a constant external magnetic field to obtain the magnetic particle-protein fiber assembly.

[0014] Preferably, the magnetic nanoparticles include one or more of Fe3O4 nanoparticles, γ-Fe2O3 nanoparticles, CoFe2O4 nanoparticles, and Prussian blue nanoparticles;

[0015] The magnetic nanoparticles have a particle size of 1–300 nm.

[0016] Preferably, the negatively charged polymer includes one or more of polydopamine, chitosan, and hydroxymethyl cellulose;

[0017] The coating temperature is room temperature, and the time is ≥8h;

[0018] The copper ion loading time is ≥12h;

[0019] The functional proteins include one or more of fibrinogen, albumin, collagen, fibronectin, laminin, and cytokines.

[0020] The incubation time is 10–20 minutes;

[0021] The external magnetic field used for the magnetic attraction is provided by the magnetic frame, and the magnetic attraction time is 2 to 5 minutes.

[0022] Preferably, the magnetic field strength of the constant external magnetic field used for directional assembly is 0.5 to 1 mT, and the directional assembly time is 10 to 30 min.

[0023] The present invention provides a magnetic particle-protein fiber assembly prepared by the preparation method described above, comprising oriented colloidal fibers, wherein the colloidal fibers include a backbone and functional proteins covering the surface of the backbone, and the backbone is formed by coated magnetic nanoparticles loaded with copper ions.

[0024] This invention provides the application of the magnetic particle-protein fiber assembly described above in the preparation of scarless wound healing materials.

[0025] This invention provides a method for preparing a magnetic particle-protein fiber dressing, comprising the following steps:

[0026] The intermediate assembly of magnetic composite microspheres was obtained according to the preparation method described in the above technical solution;

[0027] The resuspended solution of the intermediate assembly of the magnetic composite microspheres was transferred to the surface of the substrate, and then oriented assembly was carried out under the control of a constant external magnetic field to obtain the magnetic particle-protein fiber dressing.

[0028] Preferably, the substrate comprises one or more of methacrylamide gelatin hydrogel, methacrylamide hyaluronic acid hydrogel, sodium alginate hydrogel, and PEG hydrogel;

[0029] The magnetic field strength of the constant external magnetic field used in the directional assembly is 0.5 to 1 mT, and the directional assembly time is 10 to 30 min.

[0030] This invention provides a magnetic particle-protein fiber dressing prepared by the preparation method described above, comprising a substrate and a magnetic particle-protein fiber assembly disposed on the surface of the substrate;

[0031] The magnetic particle-protein fiber assembly comprises oriented colloidal fibers, each comprising a backbone and functional proteins covering the surface of the backbone, the backbone being formed from copper-loaded coated magnetic nanoparticles.

[0032] This invention provides the application of the magnetic particle-protein fiber dressing described above in the preparation of medical devices for scarless wound healing.

[0033] This invention provides a method for preparing a magnetic particle-protein fiber assembly, comprising the following steps: coating magnetic nanoparticles to obtain coated magnetic nanoparticles, wherein the coating material of the coated magnetic nanoparticles is a negatively charged polymer; mixing the coated magnetic nanoparticles, water-soluble copper salt, and water to load copper ions, thereby obtaining copper-loaded coated magnetic nanoparticles; mixing the copper-loaded coated magnetic nanoparticles, functional protein, and buffer solution for incubation to obtain a magnetic microsphere-protein complex solution; placing the magnetic microsphere-protein complex solution in an external magnetic field for magnetic adsorption to obtain a magnetic composite microsphere intermediate assembly; and directionally assembling the resuspension of the magnetic composite microsphere intermediate assembly under the control of a constant external magnetic field to obtain a magnetic particle-protein fiber assembly. This invention first uses a negatively charged polymer as a coating material to coat magnetic nanoparticles, resulting in a negatively charged polymer coating layer that forms core-shell structured coated magnetic nanoparticles. Then, copper ions are loaded onto the surface of the negatively charged polymer coating layer through electrostatic adsorption, resulting in copper-loaded coated magnetic nanoparticles. The magnetic microsphere-protein complex formed by incubating the copper-loaded coated magnetic nanoparticles and functional proteins is then subjected to an external magnetic field to form a magnetic composite microsphere intermediate assembly. This intermediate assembly takes the form of colloidal fibers (containing shorter fibers), with the functional protein uniformly covering the surface of the copper-loaded coated magnetic nanoparticle framework. Finally, by controlling the application of a constant external magnetic field, the colloidal fibers are oriented and grown along the magnetic field lines, resulting in a magnetic particle-protein fiber assembly. The preparation method provided by this invention can easily achieve the directional arrangement of colloidal fibers by controlling the magnetic field. At the same time, the functional protein and copper-loaded coated magnetic nanoparticle skeleton structure of the obtained colloidal fibers have high stability, which simplifies the operation process and improves the therapeutic efficiency of the obtained magnetic particle-protein fiber assembly.

[0034] This invention provides a magnetic particle-protein fiber assembly prepared by the preparation method described above, comprising oriented colloidal fibers. The colloidal fibers include a backbone and functional proteins covering the surface of the backbone. The backbone is formed by coated magnetic nanoparticles loaded with copper ions. Targeting the three key stages of skin damage repair—bacterial infection, proliferation, and remodeling—this invention provides a magnetic particle-protein fiber assembly capable of achieving rapid, scar-free, full-coverage repair across all three stages. The basic backbone and assembly units of the magnetic particle-protein fiber assembly provided by this invention utilize negatively charged polymer-functionalized magnetic nanoparticles with copper ion affinity (specifically, Fe3O4@PDA-Cu magnetic nanoparticles in this embodiment), which combine with functional proteins to form colloidal fibers that can be oriented under magnetic field induction, thereby constructing the magnetic particle-protein fiber assembly. First, in the first stage (bacterial infection stage), the framework formed by copper-loaded coated magnetic nanoparticles exhibits excellent photothermal conversion performance and copper ion release capacity. Through the synergistic effect of photothermal and copper ion release, it can reduce bacterial inflammation, effectively eliminate bacteria, help reduce the risk of wound infection, and accelerate the wound healing process. Simultaneously, the copper-loaded coated magnetic nanoparticles possess good biocompatibility, reducing the irritation and damage to tissues caused by the assembly material, thus improving the safety and effectiveness of treatment. Second, in the second stage (proliferation stage) and the third stage (remodeling stage), the directional arrangement of the magnetic particle-protein fiber assembly provided by this invention provides an orderly growth environment for cells and tissues, enabling precise regulation of cell migration, proliferation, and remodeling processes. This effectively avoids abnormal behavior of fibroblasts during the repair process, thereby reducing scar formation while ensuring rapid wound healing. Furthermore, the copper ions in the framework formed by the copper-loaded coated magnetic nanoparticles can be continuously released within 28 days, catalyzing the generation of nitric oxide (NO) from endogenous nitrosothiols (RSNO), further promoting scarless skin repair and restoring the integrity of skin function.

[0035] In summary, the magnetic particle-protein fiber assembly provided by this invention has good biocompatibility and stability, and can play a synergistic role in different stages of tissue repair. At the same time, the application of multifunctional gas molecules NO to inhibit fibroblast differentiation can significantly improve the quality of wound healing, reduce scar formation, and restore the aesthetics and function of the skin.

[0036] This invention provides a magnetic particle-protein fiber dressing prepared by the preparation method described above, comprising a substrate and a magnetic particle-protein fiber assembly disposed on the surface of the substrate; the magnetic particle-protein fiber assembly comprises directionally aligned colloidal fibers, each colloidal fiber comprising a backbone and functional proteins covering the surface of the backbone, the backbone being formed by copper-ion-loaded coated magnetic nanoparticles. The magnetic particle-protein fiber dressing provided by this invention is used in wound healing medical devices and can be directly applied to wound healing. Attached Figure Description

[0037] Figure 1 This is a typical transmission electron microscope image of Fe3O4@PDA-Cu;

[0038] Figure 2 X-ray photoelectron spectroscopy (XPS) diagram of Fe3O4@PDA-Cu;

[0039] Figure 3 Infrared thermal images of Fe3O4@PDA-Cu with different concentrations under near-infrared laser irradiation;

[0040] Figure 4 The TGA thermal analysis diagram of the assembly of Fe3O4@PDA-Cu and fibrinogen;

[0041] Figure 5 Typical transmission electron microscope image of intermediate products in assembly;

[0042] Figure 6 A typical scanning electron microscope image of multifunctional magnetic particles-protein fibers;

[0043] Figure 7 The cumulative / non-cumulative release of copper ions from multifunctional magnetic particles-protein fibers over 28 days;

[0044] Figure 8 The amount of NO generated within 14 days for multifunctional magnetic particles-protein fibers;

[0045] Figure 9 This is a multifunctional magnetic particle-protein fiber dressing;

[0046] Figure 10 The therapeutic effect of a multifunctional magnetic particle-protein fiber dressing on infected wounds in mice;

[0047] Figure 11 Staining of healed skin tissue sections from mice on day 14. Detailed Implementation

[0048] This invention provides a method for preparing a magnetic particle-protein fiber assembly, comprising the following steps:

[0049] Magnetic nanoparticles are coated to obtain coated magnetic nanoparticles, wherein the coating material of the coated magnetic nanoparticles is a negatively charged polymer.

[0050] The coated magnetic nanoparticles, water-soluble copper salt, and water were mixed to load copper ions, thereby obtaining coated magnetic nanoparticles loaded with copper ions.

[0051] The copper-loaded coated magnetic nanoparticles, functional protein and buffer solution were mixed and incubated to obtain a magnetic microsphere-protein complex solution.

[0052] The magnetic microsphere-protein complex solution was placed in an external magnetic field for magnetic attraction to obtain an intermediate assembly of magnetic composite microspheres.

[0053] The magnetic composite microsphere intermediate assembly was oriented and assembled under the control of a constant external magnetic field to obtain the magnetic particle-protein fiber assembly.

[0054] In this invention, unless otherwise specified, all raw materials / components used in the preparation are commercially available products well known to those skilled in the art.

[0055] This invention involves coating magnetic nanoparticles to obtain coated magnetic nanoparticles, wherein the coating material is a negatively charged polymer. In this invention, the coated magnetic nanoparticles comprise magnetic nanoparticles and a coating material covering the surface of the magnetic nanoparticles. Preferably, the magnetic nanoparticles include one or more of Fe3O4 nanoparticles, γ-Fe2O3 nanoparticles, CoFe2O4 nanoparticles, and Prussian blue nanoparticles. In a specific embodiment of this invention, the magnetic nanoparticles are Fe3O4 nanoparticles. The particle size of the magnetic nanoparticles is preferably 1–300 nm.

[0056] In a specific embodiment of the present invention, when the magnetic nanoparticles are specifically selected as Fe3O4 nanoparticles, the preparation method of the Fe3O4 nanoparticles preferably includes the following steps: mixing an inorganic water-soluble iron salt, polyacrylic acid, urea, and an alcohol solvent and carrying out a solvothermal reaction to obtain Fe3O4 nanoparticles. The inorganic water-soluble iron salt is preferably one or more of ferric chloride, ferric nitrate, and ferric sulfate. The alcohol solvent is preferably ethylene glycol. The mass ratio of the inorganic water-soluble iron salt, polyacrylic acid, and urea is preferably (0.8–0.85):(0.25–0.3):(1.5–2), specifically 0.81:0.29:1.8. The present invention does not have special requirements for the amount of alcohol solvent used. The mixing of the inorganic water-soluble iron salt, polyacrylic acid, urea, and alcohol solvent is preferably carried out under ultrasonic conditions, and the solvothermal reaction is preferably carried out in a stainless steel reactor with a polytetrafluoroethylene liner. The temperature of the solvothermal reaction is preferably 200–300°C. The time of the solvothermal reaction is preferably ≥12 h. The solid product obtained from the hydrothermal reaction is preferably washed sequentially with ethanol and then with water to obtain Fe3O4 nanoparticles. The ethanol washing is preferably performed under ultrasonic conditions. The water washing is preferably performed with ultrapure water or deionized water, and is preferably performed under ultrasonic conditions. The number of water washings is preferably 3 to 5 times. The ethanol washing time is preferably 5 to 10 minutes, and the time for each water washing is preferably 5 to 10 minutes. In this invention, the obtained Fe3O4 nanoparticles are dispersed in deionized water and stored at 4°C.

[0057] In this invention, the coating material is a loaded polymer. In this invention, the negatively charged polymer includes one or more of polydopamine, chitosan, and hydroxymethyl cellulose. In a specific embodiment of this invention, the negatively charged polymer is specifically polydopamine.

[0058] In this invention, when the negatively charged polymer is specifically chitosan and / or hydroxymethyl cellulose, it is preferred that the magnetic nanoparticles be directly coated with chitosan and / or hydroxymethyl cellulose in a solution environment. This invention does not have special requirements for the specific implementation process of the coating with chitosan and / or hydroxymethyl cellulose.

[0059] In this invention, when the negatively charged polymer is specifically polydopamine, the coating process preferably includes the following steps:

[0060] Magnetic nanoparticles, dopamine, and buffer solution are mixed and polymerized under alkaline conditions to obtain coated magnetic nanoparticles. The coating material for the coated magnetic nanoparticles is polydopamine. In this invention, the mass ratio of Fe3O4 nanoparticles to dopamine is preferably 3:(1-3). The buffer solution is preferably Tris-HCl, with a pH of 8.5 and a molar concentration of 10 mM. The mixing of the magnetic nanoparticles, polymeric monomer, and buffer solution is preferably carried out under ultrasonic conditions. The polymerization coating temperature is preferably room temperature, and the polymerization coating time is preferably ≥8 hours.

[0061] In a specific embodiment of the present invention, when the polymeric monomer is dopamine (DA), the present invention obtains a polydopamine (PDA) coating layer by oxidative polymerization of dopamine on the carboxyl surface of Fe3O4 under alkaline conditions, forming a core-shell structure Fe3O4@PDA coating material.

[0062] In this invention, the solid product obtained by polymerization and coating is preferably subjected to ethanol washing and water washing sequentially to obtain coated magnetic nanoparticles. The ethanol washing is preferably performed under ultrasonic conditions. The water washing is preferably performed with ultrapure water or deionized water, and is preferably performed under ultrasonic conditions. The number of water washings is preferably 3 to 5 times. The ethanol washing time is preferably 5 to 10 minutes, and the time for each water washing is preferably 5 to 10 minutes.

[0063] After obtaining the coated magnetic nanoparticles, the present invention mixes the coated magnetic nanoparticles, a water-soluble copper salt, and water to load copper ions, thereby obtaining coated magnetic nanoparticles loaded with copper ions. In the present invention, the water-soluble copper salt is preferably one or more of copper chloride, copper nitrate, and copper sulfate. The water is preferably deionized water. In a specific embodiment of the present invention, the water-soluble copper salt is specifically preferably CuCl2·2H2O. The mixing of the coated magnetic nanoparticles, the water-soluble copper salt, and the water preferably includes: dissolving the water-soluble copper salt in water to obtain a copper salt solution; and dispersing the coated magnetic nanoparticles in the copper salt solution. The molar concentration of the copper salt solution is preferably 1 mM. The dispersion is preferably carried out under ultrasonic conditions. The temperature for copper ion loading is preferably room temperature, and the time for copper ion loading is preferably ≥12 h. The copper ion loading is preferably carried out under stirring conditions.

[0064] In a specific embodiment of the present invention, the present invention first obtains a polydopamine (PDA) coating layer by oxidative polymerization of dopamine on the carboxyl surface of Fe3O4 under alkaline conditions, forming a core-shell structure Fe3O4@PDA coating material; then copper ions complex with the catecholamine groups of PDA to form Fe3O4@PDA-Cu magnetic nanoparticles through non-covalent interactions.

[0065] In this invention, the solid product obtained by copper ion loading is preferably washed with water to obtain copper ion-coated magnetic nanoparticles. The washing is preferably performed with ultrapure water or deionized water, preferably under ultrasonic conditions, and the number of washes is preferably 3 to 5. The duration of each wash is preferably 5 to 10 minutes.

[0066] After obtaining copper-loaded coated magnetic nanoparticles, the present invention mixes and incubates the copper-loaded coated magnetic nanoparticles, functional protein, and buffer solution to obtain a magnetic microsphere-protein complex solution. In this invention, the functional protein preferably includes one or more of fibrinogen, albumin, collagen, fibronectin, laminin, and cytokines. In this invention, the cytokines are a mixture of proteins secreted by cells, and a general method for obtaining cytokines is to culture adherent cells for a certain period of time and then mix the supernatant with magnetic beads. In a specific embodiment of this invention, the functional protein is specifically fibrinogen. The buffer solution is preferably PBS. The mixing of the copper-loaded coated magnetic nanoparticles, functional protein, and buffer solution preferably includes: dissolving the functional protein in the buffer solution to obtain a protein solution; and mixing the protein solution with the solvent of the copper-loaded coated magnetic nanoparticles. The mass concentration of the protein solution is preferably 0.1 mg / mL. The incubation is preferably carried out under slow shaking conditions, the incubation temperature is preferably 1–4 °C, and the incubation time is preferably 10–20 min.

[0067] After obtaining the magnetic microsphere-protein complex solution, the present invention places the magnetic microsphere-protein complex solution in an external magnetic field for magnetic attraction to obtain an intermediate assembly of magnetic composite microspheres. In the present invention, the external magnetic field used for magnetic attraction is preferably provided by a magnetic rack. In a specific embodiment of the present invention, the magnetic microsphere-protein complex solution is preferably placed on the magnetic rack and left to stand. The magnetic attraction time is preferably 2 to 5 minutes. The magnetic attraction is preferably carried out under standing conditions. After the magnetic attraction is completed, black agglomerates are formed on the wall of the container, and the external magnetic field is removed.

[0068] Preferably, the present invention involves resuspending the intermediate assembly of the magnetic composite microspheres obtained by magnetic attraction from the wall of a vessel to obtain a resuspension of the intermediate assembly of the magnetic composite microspheres, and then performing subsequent directional assembly on the resuspension of the intermediate assembly of the magnetic composite microspheres.

[0069] After obtaining the intermediate magnetic composite microsphere assembly, the resuspension of the intermediate magnetic composite microsphere assembly is directionally assembled under the control of a constant external magnetic field to obtain a magnetic particle-protein fiber assembly. In this invention, the preferred method for setting the constant external magnetic field is to place magnets on both sides or one side of the container containing the resuspension of the intermediate magnetic composite microsphere assembly. Specifically, the preferred methods for setting the constant external magnetic field are the first and second cases. First case: A permanent magnet with the same magnetic flux is placed parallel to each other on both sides of the container containing the resuspension of the intermediate magnetic composite microsphere assembly in any direction, ensuring that the material in the resuspension does not experience magnetic surge. Second case: A magnet is placed in any direction of the container containing the resuspension of the intermediate magnetic composite microsphere assembly, and the distance between the magnet and the container is set such that a strong magnetic surge does not occur in the material in the resuspension. In this invention, in the second scenario, the magnet is preferably an 80 mT magnet, and the distance between the magnet and the container holding the resuspended liquid containing the magnetic composite microsphere intermediate assembly is 2.5 cm. In both of the above-mentioned constant external magnetic field configurations, this invention can form oriented assemblies with the orientation direction parallel to the magnetic field direction. In this invention, the magnetic field strength of the constant external magnetic field used for oriented assembly is 0.5–1 mT, and the oriented assembly time is 10–30 min. The oriented assembly is preferably performed under static conditions. This invention preferably achieves effective binding stability between the functional protein in the colloidal fiber and the framework formed by the copper-loaded coated magnetic nanoparticles through oriented assembly, while also facilitating the growth and oriented assembly of the colloidal fiber.

[0070] The present invention provides a magnetic particle-protein fiber assembly prepared by the preparation method described above, comprising oriented colloidal fibers, wherein the colloidal fibers include a backbone and functional proteins covering the surface of the backbone, and the backbone is formed by coated magnetic nanoparticles loaded with copper ions.

[0071] This invention provides the application of the magnetic particle-protein fiber assembly described above in the preparation of scarless wound healing materials.

[0072] This invention provides a method for preparing a magnetic particle-protein fiber dressing, comprising the following steps:

[0073] The intermediate assembly of magnetic composite microspheres was obtained according to the preparation method described in the above technical solution;

[0074] The resuspended solution of the intermediate assembly of the magnetic composite microspheres was transferred to the surface of the substrate, and then oriented assembly was carried out under the control of a constant external magnetic field to obtain the magnetic particle-protein fiber dressing.

[0075] The present invention obtains an intermediate assembly of magnetic composite microspheres according to the preparation method described above. The preparation process of the intermediate assembly of magnetic composite microspheres will not be described in detail here.

[0076] After obtaining the intermediate assembly of magnetic composite microspheres, the present invention transfers the resuspension of the intermediate assembly to the surface of a substrate and performs directional assembly under the control of a constant external magnetic field to obtain the magnetic particle-protein fiber dressing. In this invention, the substrate preferably includes one or more of methacrylamide gelatin hydrogel, methacrylamide hyaluronic acid hydrogel, sodium alginate hydrogel, and PEG hydrogel. In this invention, the PEG hydrogel is specifically poly(ethylene glycol) diacrylate.

[0077] The magnetic field strength of the constant external magnetic field used for directional assembly is preferably 0.5 to 1 mT, and the directional assembly time is preferably 10 to 30 min.

[0078] In a specific embodiment of the present invention, the preparation method of the methacrylamide gelatin hydrogel preferably includes the following steps: dissolving methacrylamide gelatin (GelMA) powder in a photocrosslinking agent solution to obtain a hydrogel precursor solution; subjecting the hydrogel precursor solution to a crosslinking reaction under ultraviolet light irradiation to obtain the methacrylamide gelatin hydrogel. The photocrosslinking agent solution comprises a photocrosslinking agent and PBS buffer, and the photocrosslinking agent is preferably lithium phenyl-2,4,6-trimethylbenzoylphosphine. The mass ratio of the GelMA powder to the photocrosslinking agent is preferably 10:12.5. The wavelength of the ultraviolet light is preferably 405 nm. The crosslinking reaction time is preferably 1 min.

[0079] This invention provides a magnetic particle-protein fiber dressing prepared by the preparation method described above, comprising a substrate and a magnetic particle-protein fiber assembly disposed on the surface of the substrate;

[0080] The magnetic particle-protein fiber assembly comprises oriented colloidal fibers, each comprising a backbone and functional proteins covering the surface of the backbone, the backbone being formed from copper-loaded coated magnetic nanoparticles.

[0081] This invention provides the application of the magnetic particle-protein fiber dressing described above in the preparation of medical devices for scarless wound healing.

[0082] The magnetic particle-protein fiber dressing provided by this invention targets the first stage of tissue repair, where functionalized nanoparticles reduce bacterial inflammation through the synergistic effect of photothermal and copper ions. For the second and third stages, the directionally aligned colloidal fibers induce fibroblasts and collagen to arrange themselves in an orderly spatial order. Furthermore, over a period of 28 days, copper ions are continuously released and catalyze the generation of NO from endogenous RSNO, synergistically promoting scarless skin repair and restoring the integrity of skin function.

[0083] In a specific embodiment of the present invention, the method for preparing the magnetic particle-protein fiber assembly provided by the present invention includes the following steps: mixing Fe3O4 nanoparticles, dopamine, and buffer solution, and polymerizing and coating them under alkaline conditions to obtain Fe3O4@PDA coated material; mixing the Fe3O4@PDA coated material, water-soluble copper salt, and water for copper ion loading to obtain Fe3O4@PDA-Cu magnetic nanoparticles; mixing the Fe3O4@PDA-Cu magnetic nanoparticles, fibrinogen, water, and buffer solution for incubation to obtain a magnetic microsphere-protein complex solution; placing the magnetic microsphere-protein complex solution in an external magnetic field for magnetic adsorption to obtain a magnetic composite microsphere intermediate assembly; and directionally assembling the resuspension of the magnetic composite microsphere intermediate assembly under the control of a constant external magnetic field to obtain the magnetic particle-protein fiber assembly. This invention first involves the oxidative polymerization of dopamine on the carboxyl surface of Fe3O4 under alkaline conditions to obtain a polydopamine (PDA) coating layer, forming a core-shell structured Fe3O4@PDA coated material. Then, copper ions complex with the catecholamine groups of PDA, forming Fe3O4@PDA-Cu magnetic nanoparticles through non-covalent interactions. The magnetic microsphere-protein complex formed by incubating the Fe3O4@PDA-Cu magnetic nanoparticles and fibrinogen is then subjected to an external magnetic field to form a magnetic composite microsphere intermediate assembly. This intermediate assembly takes the form of colloidal fibers, with fibrinogen uniformly covering the surface of the Fe3O4@PDA-Cu magnetic nanoparticle framework. Finally, by controlling the static magnetic field, the colloidal fibers can be oriented along the magnetic field lines to obtain a magnetic particle-protein fiber assembly. The preparation method provided by this invention allows for convenient orientation of the colloidal fibers through magnetic field control, simplifying the operation process and improving the therapeutic efficiency of the obtained magnetic particle-protein fiber assembly.

[0084] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0085] Example 1

[0086] (1) Accurately weigh 0.81g ferric chloride, 0.29g polyacrylic acid, and 1.8g urea. Disperse the above reagents in 30mL ethylene glycol. After thorough dissolution by ultrasonic stirring, transfer to a reaction vessel and heat in a high-temperature oven at 200℃ for 12h. Superparamagnetic Fe3O4 nanoparticle cores are prepared by a one-pot hydrothermal synthesis method. After the reaction, wait for the reaction vessel to cool to room temperature. Disperse the obtained black precipitate in ethanol by ultrasonication for 5-10min. After magnetic separation, redisperse the precipitate in deionized water and sonicate for 5-10min. Separate by magnetic attraction using an external magnetic field. Wash repeatedly with deionized water 3-5 times. Finally, disperse in deionized water to obtain Fe3O4 superparamagnetic cores with a particle size of 100nm. The transmission electron microscope image of the Fe3O4 superparamagnetic cores prepared in this example is shown below. Figure 1 As shown. The X-ray photoelectron spectroscopy (XPS) pattern of the Fe3O4 superparamagnetic core prepared in this embodiment is shown in the figure. Figure 2 As shown.

[0087] (2) Take 30 mg of the product from step (1) and disperse it in 120 mL of Tris-HCl (10 mM, pH = 8.5). After sonication for 30 min, add 30 mg of DA and sonicate to dissolve it completely. Stir the mixture continuously at 200 rpm for 8 h at room temperature. After the reaction is complete, disperse the obtained product in ethanol and sonicate for 5-10 min. After magnetic separation, redisperse the precipitate in deionized water and sonicate for 5-10 min. Separate it using an external magnetic field and wash it repeatedly with deionized water 3-5 times. Finally, disperse it in deionized water to obtain Fe3O4@PDA with a hydrodynamic diameter of about 150 nm.

[0088] (3) Add 6.82 mg CuCl2·2H2O to 40 mL of deionized water, and ultrasonically disperse the product from step (2) in CuCl2 solution. Stir at room temperature for 12 h. Separate the product, collect it with a magnet, and wash it three times with ultrapure water to obtain Fe3O4@PDA-Cu. Figure 3 Infrared thermal images of Fe3O4@PDA-Cu at different concentrations under near-infrared laser irradiation were prepared using PBS buffer.

[0089] (4) Accurately weigh 1 mg of fibrinogen and place it in a centrifuge tube. Add 1 mL of PBS solution to prepare a protein solution (protein concentration 1 mg / mL). Dilute the protein solution with PBS to 0.1 mg / mL. Take an aqueous dispersion containing 100 μg of Fe3O4@PDA-Cu prepared in step (3) and add it to the protein solution. Gently invert the centrifuge tube and incubate at 4°C or room temperature for 10 min to obtain a PBS liquid phase system of magnetic microsphere-protein complex. The product does not need to be washed and can be used for later use.

[0090] (5) Place the PBS liquid phase system of the magnetic microsphere-protein complex from step (4) on a magnetic rack and let it stand for 2 minutes. After black aggregates form on the wall of the container, remove the external magnetic field. Gently resuspend the aggregates by pipetting with a pipette tip and transfer them to a well plate with a coverslip at the bottom (the coverslip is placed to facilitate direct characterization of the assembly morphology later). The transmission electron microscope image of the obtained magnetic composite microsphere intermediate assembly is shown below. Figure 5 As shown. A constant external magnetic field of approximately 0.5 mT was applied to both sides of the well plate, and the reaction was allowed to proceed statically for 10 min, resulting in magnetic particle-protein fiber assemblies on the coverslip surface. The coverslip at the bottom was slowly removed with tweezers, allowing direct observation of the morphology and size of the magnetic particle-protein fiber assemblies under an optical microscope. Under typical conditions, the magnetic particle-protein fiber assemblies exhibit a linear structure, as shown... Figure 6 As shown, the diameter of a single fiber is 2μm, and the diameter of a cross-linked fiber is 15μm. Figure 4 The image shows a TGA thermal analysis of the magnetic particle-protein fiber assembly prepared in this embodiment. Figure 6 Scanning electron microscope image of the magnetic particle-protein fiber assembly prepared in this embodiment.

[0091] Example 2

[0092] (1) Preparation of hydrogel dressing: Take 20 mL of PBS buffer and add 50 mg of phenyl-2,4,6-trimethylbenzoyl lithium phosphine. Heat the above solution at 50 °C for 15 min and store at 4 °C for later use. Weigh 10 mg of dried GelMA powder and dissolve it in 5 mL of the previously prepared phenyl-2,4,6-trimethylbenzoyl lithium phosphine solution, and heat at 60 °C for 30 min to obtain the hydrogel precursor solution. Irradiate the hydrogel precursor solution with 405 nm ultraviolet light for 1 min to obtain the hydrogel dressing.

[0093] (2) Preparation of oriented multifunctional magnetic particle-protein fiber dressing: The hydrogel dressing obtained in step (1) was placed at the bottom of a 24-well plate. The PBS liquid phase system of the magnetic microsphere-protein complex obtained in step (4) of Example 1 was placed on a magnetic rack and left to stand for 2 min. After black agglomerates formed on the wall of the vessel, the external magnetic field was removed. The agglomerates were gently resuspended by pipetting and transferred to the well plate with the hydrogel dressing at the bottom (the hydrogel dressing was placed to facilitate subsequent in vivo application). A constant external magnetic field of about 0.5 mT was applied to both sides of the well plate and allowed to react for 30 min, thus obtaining magnetic particle-protein fiber assemblies on the surface of the hydrogel, and thus obtaining the magnetic particle-protein fiber dressing. The magnetic particle-protein fiber dressing at the bottom was slowly removed with tweezers. Figure 9 This is a photograph of the multifunctional magnetic particle-protein fiber dressing prepared in this embodiment.

[0094] Comparative Example 1

[0095] Magnetic nanoparticle-protein self-assembly: The hydrogel dressing obtained in step (1) of Example 2 was placed at the bottom of a 24-well plate. The PBS liquid phase system of the magnetic microsphere-protein complex obtained in step (4) of Example 1 was placed on a magnetic rack and allowed to stand for 2 min. After black agglomerates formed on the wall of the vessel, the external magnetic field was removed. The agglomerates were then gently resuspended by pipetting and transferred to the well plate with the hydrogel dressing at the bottom. After standing for 30 min, non-oriented magnetic particles-protein fibers were formed on the surface of the hydrogel.

[0096] Comparative Example 1 shows that electrostatic self-assembly can also form fibers on the surface of hydrogels, but the network is intricate and lacks directional morphology, which does not conform to the growth habits of fibroblasts in skin repair. Although the product prepared in Comparative Example 1 shows a certain fiber orientation in electron micrographs, the fibers are not completely straight, and different columns intersect to form a network structure. In Example 2 of this invention, the present invention uses a magnetic field to intervene in the directional assembly process on the surface of the substrate material. The resulting dressing product consists of fully oriented assembled fibers that are almost completely parallel. This is because, in the absence of an external magnetic field (Comparative Example 1), the Earth's magnetic field interferes with self-assembly, but due to the relatively weak magnetic field, the obtained fibers have a certain degree of alignment. When the magnetic field is increased to the range described in Example 2, the magnetic field can guide the assembled material to align along the direction of the magnetic field, resulting in a higher degree of alignment. In summary, compared with Comparative Example 1, the magnetic protein fibers prepared in the embodiments of this invention have a higher degree of alignment.

[0097] Example 3

[0098] (1) In the in vitro experiment, in order to monitor the copper ion release capacity of the multifunctional magnetic particle-protein fiber dressing prepared in Example 2, the multifunctional magnetic particle-protein fiber dressing was immersed in 1 mL PBS. The PBS solution was collected on days 1, 3, 6, 9, 14, 21 and 28 for inductively coupled plasma mass spectrometry (ICP-MS) testing. Fresh PBS was added immediately after each collection. Figure 7 The cumulative / non-cumulative release of copper ions over 28 days was measured in the multifunctional magnetic particle-protein fiber dressing prepared in Example 2. Figure 7 It can be seen that the multifunctional magnetic particle-protein fiber dressing prepared in Example 2 can continuously release copper ions for 28 days.

[0099] (2) Monitoring of NO catalytic generation capacity: The operation method is similar to that in step (1). The multifunctional magnetic particle-protein fiber dressing is soaked in 1 mL PBS. NO donors (10 μM S-nitroso-N-acetylpenicillamine and 10 μM reduced glutathione) are added every 6 h. The amount of NO generated is detected by the classic Griess Reagent method for 14 consecutive days. Figure 8 The NO generation within 14 days is the amount of the multifunctional magnetic particle-protein fiber dressing prepared in Example 2.

[0100] Example 4

[0101] (1) For in vivo experiments, 10 BALB / C mice (n=5 per group) were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. to verify the therapeutic effect on skin wounds. The mice were housed in an SPF-grade environment at the experimental animal center. Male BALB / C mice (6-8 weeks old) were housed separately for one week with free access to food and water. The mice were anesthetized with 3% isoflurane for at least 5 minutes using a small animal anesthesia system. Hair was removed from the back of the mice with electric clippers, and hair removal cream was applied with cotton swabs. General anesthesia was continued as described above to ensure that the mice would not wake up during the subsequent surgery. The skin was wiped with 70% ethanol. After drying, the skin was rinsed with PBS. Two full-thickness wounds were made on the dorsal side of both sides of the midline using an 8 mm biopsy punch, extending through the sarcolemma. 100 μL of a mixed suspension of Staphylococcus aureus and Escherichia coli (1×10⁻⁶) was applied to the wound site. 8 (CFU / mL)

[0102] (2) Wound healing analysis: Skin repair materials were placed on the back wounds of mice (control group and magnetic particle-protein fiber dressing group prepared in Example 2, respectively). A circular reference was prepared and placed next to the wound. Wound photographs were taken with a digital camera on days 0, 3, 7, and 14 of the experiment, until the wound was completely closed. After the experiment, the mice were anesthetized with 3% isoflurane for at least 5 minutes and euthanized by cervical dislocation. Paraffin sections and HE staining were performed on the healed skin wounds of the mice. Figure 10 The therapeutic effect of a multifunctional magnetic particle-protein fiber dressing on infected wounds in mice; Figure 11 Staining of healed skin tissue sections from mice on day 14. Figure 10 and Figure 11 It can be seen that, compared with the control group, the multifunctional magnetic particle-protein fiber dressing prepared in Example 2 of the present invention can achieve rapid repair of skin damage without scarring.

[0103] As demonstrated by the above embodiments, this invention can conveniently achieve the directional alignment of colloidal fibers through magnetic field control, simplifying the operation process and improving treatment efficiency. This method, by integrating directional morphology and biological cues, provides a new perspective and approach for scarless healing research. By precisely controlling the morphology and bioactivity of the material, more refined regulation is achieved during wound healing. This invention designs polydopamine-functionalized magnetic nanoparticles with copper ion affinity. This design not only endows the nanoparticles with excellent biocompatibility and stability but also gives them unique magnetic responsiveness and copper ion binding ability. This allows them to reduce bacterial inflammation through photothermal and metal ion synergistic effects, helping to reduce the risk of wound infection and accelerate the wound healing process. Polydopamine-functionalized magnetic nanoparticles have good biocompatibility, reducing material irritation and damage to tissues, and improving the safety and effectiveness of treatment. Under the induction of a magnetic field, the colloidal fibers formed by the combination of polydopamine-functionalized magnetic nanoparticles and fibrinogen can align directionally along the magnetic field lines, forming a micron-scale fibrinogen scaffold. This directional alignment provides an ordered growth environment for cells and tissues. The system designed in this invention can exert a synergistic effect at different stages of tissue repair. Applying the multifunctional gas molecule NO to inhibit fibroblast differentiation provides new ideas and insights for scarless skin repair. It can significantly improve the quality of wound healing, reduce scar formation, and restore the aesthetics and function of the skin.

[0104] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. The application of a magnetic particle-protein fiber dressing in the preparation of medical devices for scarless wound healing, characterized in that, The preparation method of the magnetic particle-protein fiber dressing includes the following steps: Magnetic nanoparticles are coated to obtain coated magnetic nanoparticles. The coating material of the coated magnetic nanoparticles is a negatively charged polymer, and the negatively charged polymer is polydopamine. The coated magnetic nanoparticles, water-soluble copper salt, and water were mixed to load copper ions, thereby obtaining coated magnetic nanoparticles loaded with copper ions. The copper-loaded coated magnetic nanoparticles, functional proteins, and buffer solutions were mixed and incubated to obtain a magnetic microsphere-protein complex solution. The functional proteins included one or more of fibrinogen, albumin, collagen, fibronectin, laminin, and cytokines. The magnetic microsphere-protein complex solution was placed in an external magnetic field for magnetic attraction to obtain an intermediate assembly of magnetic composite microspheres. The resuspended solution of the intermediate assembly of the magnetic composite microspheres was transferred to the surface of the substrate, and then oriented assembly was performed under the control of a constant external magnetic field to obtain the magnetic particle-protein fiber dressing; the magnetic field strength of the constant external magnetic field used for oriented assembly was 0.5~1 mT, and the oriented assembly time was 10~30 min.

2. The application according to claim 1, characterized in that, The magnetic nanoparticles include one or more of Fe3O4 nanoparticles, γ-Fe2O3 nanoparticles, CoFe2O4 nanoparticles, and Prussian blue nanoparticles. The magnetic nanoparticles have a particle size of 1~300 nm.

3. The application according to claim 1, characterized in that, The coating temperature is room temperature, and the time is ≥8 h; The copper ion loading time is ≥12 h; The incubation time is 10-20 minutes; The external magnetic field used for the magnetic attraction is provided by the magnetic frame, and the magnetic attraction time is 2~5 minutes.

4. The application according to claim 1, characterized in that, The substrate includes one or more of methacrylamide gelatin hydrogel, methacrylamide hyaluronic acid hydrogel, sodium alginate hydrogel, and PEG hydrogel.

5. The application according to claim 1 or 4, characterized in that, The magnetic particle-protein fiber dressing includes a substrate and a magnetic particle-protein fiber assembly disposed on the surface of the substrate; The magnetic particle-protein fiber assembly comprises oriented colloidal fibers, each comprising a backbone and functional proteins covering the surface of the backbone, the backbone being formed from copper-loaded coated magnetic nanoparticles.

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