Silver-loaded nanoparticle and growth factor core-shell microneedle, and preparation method and application thereof

Abstract

This invention discloses a core-shell microneedle loaded with silver nanoparticles and growth factors, its preparation method, and its application, belonging to the field of biomedical technology. The core-shell microneedle includes a backing layer and an array of needle tips disposed on the backing layer; the needle tips in the needle tip array have a core-shell structure, with the shell layer of the needle tip loaded with protein-Ag2O2 composite nanoparticles and the core layer of the needle tip loaded with growth factors. The preparation method of the protein-Ag2O2 composite nanoparticles is as follows: a protein aqueous solution is mixed with a silver nitrate solution, and under alkaline conditions, it is reacted with a hydrogen peroxide solution to obtain the protein-Ag2O2 composite nanoparticles. The core-shell microneedles prepared by this invention, through hierarchical loading of silver peroxide nanoparticles and growth factors, respond to the microenvironment of infected skin wounds and perform programmed release, effectively preventing bacterial infection, promoting cell proliferation and angiogenesis, and driving tissue repair, effectively solving the problem of stagnant healing of infected wounds.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a core-shell microneedle loaded with silver nanoparticles and growth factors, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is a long-term metabolic disorder characterized by persistent hyperglycemia. Normal wound healing involves four stages: hemostasis, inflammation, proliferation, and remodeling, with immune regulation playing a crucial role. However, in diabetic wounds, the hyperglycemic environment inhibits fibroblast proliferation, migration, and collagen synthesis, hindering angiogenesis and thus delaying wound healing. Simultaneously, persistently high blood sugar levels promote bacterial growth, causing the healing process to stall at the inflammatory stage and inducing cell damage and apoptosis. Therefore, timely elimination of bacteria and promotion of cell proliferation and angiogenesis are effective strategies for treating diabetic wounds.

[0003] Silver peroxide nanoparticles (Ag2O2NPs) possess acid-responsive degradation properties, enabling them to simultaneously release reactive oxygen species and Ag. + In achieving the same level as traditional Ag + Ag2O2 NPs offer significantly lower required dosages for achieving the same antibacterial effect, resulting in lower cytotoxicity and making them a promising antibacterial agent. Currently, most Ag2O2 synthesis relies on polyvinylpyrrolidone (PVP) dispersion, but this method requires multiple centrifugations to collect nanoparticles, leading to low yields, easy aggregation, and poor dispersibility. Furthermore, PVP itself lacks biological activity and is used only as an inert dispersant; its coating may even delay the antibacterial process of Ag2O2. These limitations severely restrict the application of Ag2O2 in the treatment of infected wounds. Therefore, developing Ag2O2 NPs with small particle size, good dispersibility, and low cytotoxicity is of great significance.

[0004] Lactoferrin (LF) is an important component of the human immune system, possessing natural antibacterial, anti-inflammatory, and immunomodulatory biological activities. Its internal cavity structure provides binding sites for metal ions, acting as a protective layer to effectively prevent nanoparticle aggregation, thereby improving its dispersibility and stability. However, current research on the preparation of Ag2O2 nanoparticles using lactoferrin is limited, and studies on the application of lactoferrin-Ag2O2 nanoparticles in the treatment of diabetic wound healing are even rarer. Summary of the Invention

[0005] The purpose of this invention is to provide a core-shell microneedle loaded with silver nanoparticles and growth factors, its preparation method, and its application, to solve the problems existing in the prior art. The core-shell microneedle prepared by this invention, through hierarchical loading of silver peroxide nanoparticles and growth factors, responds to the microenvironment of infected skin wounds and performs programmed release, effectively preventing bacterial infection, promoting cell proliferation and angiogenesis, thereby promoting tissue repair and effectively solving the problem of stagnant healing of infected wounds.

[0006] To achieve the above objectives, the present invention provides the following solution: The present invention provides a core-shell microneedle loaded with silver nanoparticles and growth factors, the core-shell microneedle comprising a backing layer and a needle tip array disposed on the backing layer; the needle tips in the needle tip array have a core-shell structure, the shell layer of the needle tip is loaded with protein-Ag2O2 composite nanoparticles, and the core layer of the needle tip is loaded with growth factors. The preparation method of the protein-Ag2O2 composite nanoparticles includes the following steps: The protein aqueous solution was mixed with silver nitrate solution, and then reacted with hydrogen peroxide solution under alkaline conditions to obtain the protein-Ag2O2 composite nanoparticles.

[0007] Further, the concentration of the protein aqueous solution is 20 mg / mL, the concentration of the silver nitrate solution is 0.1 M, and the mass fraction of the hydrogen peroxide solution is 30%. The volume ratio of the protein aqueous solution, the silver nitrate solution, and the hydrogen peroxide solution is 5:(0.0625-0.5):(0.0065-0.052).

[0008] Furthermore, the pH of the alkaline conditions is 10-12; The mixing reaction was carried out at a temperature of 25 ± 3℃ for 20-60 min.

[0009] Optionally, the proteins include, but are not limited to, lactoferrin, serum albumin, transferrin, immunoglobulins, and hemoglobin.

[0010] Optionally, the growth factor includes, but is not limited to, recombinant fibroblast growth factor.

[0011] This invention also provides a method for preparing the above-mentioned core-shell microneedles containing silver-loaded nanoparticles and growth factors, comprising the following steps: The protein-Ag2O2 composite nanoparticles were injected into a mold to form a needle-tip shell; The growth factor is mixed with the matrix material and injected into the cavity of the needle tip shell to form the needle tip core layer; The matrix material is used to form a backing layer to obtain the core-shell microneedles.

[0012] Furthermore, the matrix material is formed by cross-linking chondroitin sulfate grafted with 3-aminophenylboronic acid with polyvinyl alcohol; The concentration of chondroitin sulfate grafted with 3-aminophenylboronic acid is 35-45 mg / mL; the concentration of polyvinyl alcohol is 90-120 mg / mL; and the volume ratio of chondroitin sulfate grafted with 3-aminophenylboronic acid to polyvinyl alcohol is 1:1-1:5.

[0013] Furthermore, after the growth factor is mixed with the matrix material, the final concentration of the growth factor is 5-20 ng / mL.

[0014] The present invention also provides the application of the above-mentioned silver-loaded nanoparticles and growth factor core-shell microneedles in the preparation of drugs for treating skin infection wounds.

[0015] Optionally, the infected skin wound includes a diabetic infected wound.

[0016] The present invention also provides a medicament for treating skin infection wounds, the medicament comprising the above-mentioned silver-loaded nanoparticles and growth factors in a core-shell microneedle.

[0017] The present invention discloses the following technical effects: This invention prepares silver peroxide nanoparticles (LF-Ag2O2NPs) templated with lactoferrin using a simple one-step biomimetic mineralization method, solving the problems of complex processes, large particle size, and poor stability associated with traditional preparation methods. LF-Ag2O2NPs achieve highly efficient antibacterial activity through the synergistic effect of LF, silver ions, and reactive oxygen species, exhibiting significant bactericidal effects against Gram-positive bacteria (Staphylococcus aureus, methicillin-resistant Staphylococcus aureus), Gram-negative bacteria (Escherichia coli), and fungi (Candida albicans). Compared to single silver ion or reactive oxygen species antibacterial strategies, LF-Ag2O2NPs offer significant advantages such as broad-spectrum antibacterial activity, significantly reduced silver usage, and decreased cytotoxicity.

[0018] The core-shell microneedles prepared in this invention use LF-Ag2O2NPs as the shell and recombinant fibroblast growth factor (bFGF) as the core. Through graded loading of LF-Ag2O2NPs and bFGF, they respond to the microenvironment of infected skin wounds and undergo programmed release. When the microneedles are applied to infected wounds, LF-Ag2O2NPs are rapidly released, decomposing in response to the slightly acidic environment of the wound and releasing silver ions and reactive oxygen species for highly effective antibacterial effects. As the bacterial infection is controlled, the microneedle core degrades in response to the microenvironment and slowly releases bFGF, continuously promoting cell proliferation and angiogenesis, thereby driving tissue repair. This programmed drug delivery system achieves a synergistic effect of multiple therapeutic benefits, including antibacterial, anti-inflammatory, angiogenesis-promoting, and epithelial remodeling effects, effectively solving the problem of stagnant healing in infected wounds. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 Transmission electron microscopy (a), particle size distribution (b), XRD pattern (c), and UV-Vis absorption spectrum (d) of LF-Ag2O2NPs prepared in Example 2, measured by TMB colorimetry. Figure 2 The results are as follows: performance test results of microneedles; where a is the SEM image of the microneedles prepared in Example 2; b is the fluorescence image of the microneedles prepared in Example 2; c is the mechanical strength of the microneedles prepared in Comparative Example 1 and Comparative Example 2; d is the drug release curve of the BSA-loaded microneedles. Figure 3 Images of bacterial colonies (a) and bacterial survival rates (b and c) after co-incubation of LF-Ag2O2NPs prepared in Example 2 with four types of bacteria. Figure 4 Images of bacterial colonies (a) and bacterial survival rates (b and c) after co-incubation of the microneedles prepared in Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3 with four types of bacteria. Figure 5 The images (a) and (b) show the cytotoxicity detection fluorescence images and cell viability statistics of the microneedles prepared in Example 2, Comparative Example 1, Comparative Example 2, and Comparative Example 3. Figure 6 The images (a) and cell migration rate (b) show the cell migration promotion ability test results of the microneedles prepared in Example 2, Comparative Example 1, and Comparative Example 3. Figure 7 Photographs of wounds treated with microneedles at different times in diabetic model mice prepared in Example 2, Comparative Example 1, and Comparative Example 3 (a), bacterial colony images collected from the exudate around the wounds in each group (b), relative wound area (c), and changes in mouse body weight (d). Figure 8 Fluorescent staining images of CD86 and CD206 in wound skin tissue of diabetic model mice 14 days after microneedle treatment prepared in Example 2, Comparative Example 1, and Comparative Example 3. Figure 9 Fluorescent staining images of CD34 and α-SMA in wound skin tissue of diabetic model mice 14 days after microneedle treatment prepared in Example 2, Comparative Example 1, and Comparative Example 3. Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0027] In the following examples, LF represents lactoferrin; Ag2O2 represents silver peroxide; CS represents chondroitin sulfate; APBA represents 3-aminophenylboronic acid; CS-APBA represents chondroitin sulfate grafted with 3-aminophenylboronic acid (grafting degree 10%-30%); MES represents morpholine ethanesulfonic acid monohydrate; EDC represents 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; NHS represents... N-Hydroxysuccinimide; bFGF represents recombinant fibroblast growth factor; PDMS represents polydimethylsiloxane. LF, AgNO3, CS, APBA, MES, EDC, and NHS were purchased from Aladdin (Shanghai, China); bFGF was purchased from novoprotein (Jiangsu, China); and PDMS was purchased from Henan MicroNano Bentech Biotechnology Co., Ltd.

[0028] S. aureus It indicates Staphylococcus aureus; E. coli It represents Escherichia coli; MRSA represents methicillin-resistant Staphylococcus aureus. C. albicans It refers to Candida albicans.

[0029] Example 1 This embodiment provides a method for preparing silver-loaded nanoparticles and growth factor core-shell microneedles: (1) Preparation of LF-Ag2O2 composite nanoparticles: 100 mg of LF was dissolved in 5 mL of deionized water and stirred at room temperature for 20 min. Then, 0.0625 mL of silver nitrate solution (0.1 M) was added and stirred at room temperature for 20 min. Next, 0.1 M sodium hydroxide solution was added dropwise until the pH of the solution was 10, and stirring was continued for 20 min. Finally, 6.5 μL of 30% hydrogen peroxide solution was added to the solution and stirred at room temperature for 20 min to obtain composite nanoparticles. The composite nanoparticles were purified by dialysis with deionized water to obtain LF-Ag2O2 composite nanoparticles.

[0030] (2) Preparation of silver-loaded nanoparticles and growth factor core-shell microneedles (LAg&FGF@CP MN): 1 g of CS was dissolved in 100 mL of MES buffer solution at pH=5, and stirred for 30 min to ensure complete dissolution. 0.12 g of EDC and 0.072 g of NHS were added, and the mixture was stirred in an ice-water bath for 1 h to activate the carboxyl groups on the CS. Then, 0.107 g of APBA was added, and the reaction proceeded for 24 h. Finally, the solution was dialyzed against deionized water for 3 consecutive days, and then lyophilized for 5 days to obtain CS-APBA.

[0031] An LF-Ag2O2 composite nanoparticle solution (100 mg / mL) was added to the surface of a PDMS mold. Vacuum was then applied to remove air bubbles, ensuring the solution filled the entire needle tip cavity. Excess liquid was then scraped off. Finally, the LF-Ag2O2 shell was obtained by drying at room temperature in a well-ventilated, dark place.

[0032] Subsequently, CS-APBA solution (40 mg / mL, grafting degree 15.8%) and polyvinyl alcohol solution (100 mg / mL) were mixed evenly at a 1:1 volume ratio to obtain CP solution; bFGF solution was added to CP solution to obtain FGF@CP solution, wherein the final concentration of bFGF was 5 ng / mL. FGF@CP solution was added to the groove of PDMS mold containing the shell, and the mold was centrifuged to ensure that FGF@CP solution fully filled the needle tip cavity.

[0033] After centrifugation, CP solution was added to form a microneedle backing layer. Finally, the microneedles were dried at room temperature, in a well-ventilated area, and protected from light. The membrane was then removed to obtain LAg&FGF@CP MN.

[0034] Example 2 This embodiment provides a method for preparing silver-loaded nanoparticles and growth factor core-shell microneedles: (1) Preparation of LF-Ag2O2 composite nanoparticles: 100 mg of LF was dissolved in 5 mL of deionized water and stirred at room temperature for 30 min. Then, 0.5 mL of silver nitrate solution (0.1 M) was added and stirred at room temperature for 30 min. Next, 0.1 M sodium hydroxide solution was added dropwise until the pH of the solution was 11, and stirring was continued for 30 min. Finally, 52 μL of 30% hydrogen peroxide solution was added to the solution and stirred at room temperature for 30 min to obtain composite nanoparticles. The composite nanoparticles were purified by dialysis with deionized water to obtain the final LF-Ag2O2 composite nanoparticles.

[0035] (2) Preparation of LAg&FGF@CP MN: 1 g of CS was dissolved in 100 mL of MES buffer solution at pH=5, and stirred for 30 min to ensure complete dissolution. 0.12 g of EDC and 0.072 g of NHS were added, and the mixture was stirred in an ice-water bath for 1 h to activate the carboxyl groups on the CS. Then, 0.107 g of APBA was added, and the reaction proceeded for 24 h. Finally, the solution was dialyzed against deionized water for 3 consecutive days, and then lyophilized for 5 days to obtain CS-APBA.

[0036] An LF-Ag2O2 composite nanoparticle solution (100 mg / mL) was added to the surface of a PDMS mold. Vacuum was then applied to remove air bubbles, ensuring the solution filled the entire needle tip cavity. Excess liquid was then scraped off. Finally, the LF-Ag2O2 shell was obtained by drying at room temperature in a well-ventilated, dark place.

[0037] Subsequently, CS-APBA solution (40 mg / mL, grafting degree 15.8%) and polyvinyl alcohol solution (100 mg / mL) were mixed thoroughly at a volume ratio of 1:3 to obtain CP solution; bFGF solution was added to CP solution to obtain FGF@CP solution, wherein the final concentration of bFGF was 10 ng / mL. FGF@CP solution was added to the groove of PDMS mold containing the shell, and the mold was centrifuged to ensure that FGF@CP solution fully filled the needle tip cavity.

[0038] After centrifugation, CP solution was added to form a microneedle backing layer. Finally, the microneedles were dried at room temperature, in a well-ventilated area, and protected from light. The membrane was then removed to obtain LAg&FGF@CP MN.

[0039] Example 3 This embodiment provides a method for preparing silver-loaded nanoparticles and growth factor core-shell microneedles: (1) Preparation of LF-Ag2O2 composite nanoparticles: 100 mg of LF was dissolved in 5 mL of deionized water and stirred at room temperature for 60 min. Then, 0.5 mL of silver nitrate solution (0.1 M) was added and stirred at room temperature for 60 min. Next, 0.1 M sodium hydroxide solution was added dropwise until the pH of the solution reached 12, and stirring was continued for 60 min. Finally, 52 μL of 30% hydrogen peroxide solution was added to the solution and stirred at room temperature for 60 min to obtain composite nanoparticles. The composite nanoparticles were purified by dialysis with deionized water to obtain the final LF-Ag2O2 composite nanoparticles.

[0040] (2) Preparation of LAg&FGF@CP MN: 1 g of CS was dissolved in 100 mL of MES buffer solution at pH=5, and stirred for 30 min to ensure complete dissolution. 0.12 g of EDC and 0.072 g of NHS were added, and the mixture was stirred in an ice-water bath for 1 h to activate the carboxyl groups on the CS. Then, 0.107 g of APBA was added, and the reaction proceeded for 24 h. Finally, the solution was dialyzed against deionized water for 3 consecutive days, and then lyophilized for 5 days to obtain CS-APBA.

[0041] An LF-Ag2O2 composite nanoparticle solution (100 mg / mL) was added to the surface of a PDMS mold. Vacuum was then applied to remove air bubbles, ensuring the solution filled the entire needle tip cavity. Excess liquid was then scraped off. Finally, the LF-Ag2O2 shell was obtained by drying at room temperature in a well-ventilated, dark place.

[0042] Subsequently, CS-APBA solution (40 mg / mL, grafting degree 15.8%) and polyvinyl alcohol solution (100 mg / mL) were mixed thoroughly at a volume ratio of 1:5 to obtain CP solution; bFGF solution was added to CP solution to obtain FGF@CP solution, wherein the final concentration of bFGF was 20 ng / mL. FGF@CP solution was added to the groove of PDMS mold containing the shell, and the mold was centrifuged to ensure that FGF@CP solution fully filled the needle tip cavity.

[0043] After centrifugation, CP solution was added to form a microneedle backing layer. Finally, the microneedles were dried at room temperature, in a well-ventilated area, and protected from light. The membrane was then removed to obtain LAg&FGF@CP MN.

[0044] Comparative Example 1 This comparative example provides a blank microneedle (CP MN), which is prepared by the following method: 1 g of CS was dissolved in 100 mL of MES buffer solution at pH=5, and stirred for 30 min to ensure complete dissolution. 0.12 g of EDC and 0.072 g of NHS were added, and the mixture was stirred in an ice-water bath for 1 h to activate the carboxyl groups on the CS. Then, 0.107 g of APBA was added, and the reaction proceeded for 24 h. Finally, the solution was dialyzed against deionized water for 3 consecutive days, and then lyophilized for 5 days to obtain CS-APBA.

[0045] CS-APBA solution (40 mg / mL, grafting degree 15.8%) and polyvinyl alcohol solution (100 mg / mL) were mixed thoroughly at a volume ratio of 1:3 to obtain CP solution. The CP solution was added to the grooves of a PDMS mold, and the mold was centrifuged to ensure the CP solution fully filled the needle tip cavity. After centrifugation, more CP solution was added to form a microneedle backing layer. Finally, the microneedles were dried at room temperature in a well-ventilated, dark place, and then demolded to obtain CP MN.

[0046] Comparative Example 2 This comparative example provides a microneedle loaded with a lactoferrin shell (LCP MN), which is prepared by the following method: 1 g of CS was dissolved in 100 mL of MES buffer solution at pH=5, and stirred for 30 min to ensure complete dissolution. 0.12 g of EDC and 0.072 g of NHS were added, and the mixture was stirred in an ice-water bath for 1 h to activate the carboxyl groups on the CS. Then, 0.107 g of APBA was added, and the reaction proceeded for 24 h. Finally, the solution was dialyzed against deionized water for 3 consecutive days, and then lyophilized for 5 days to obtain CS-APBA.

[0047] An LF solution with a concentration of 200 mg / mL was added to the surface of the PDMS mold. Vacuum was then applied to remove air bubbles, ensuring the solution filled the entire needle tip cavity. Excess liquid was then scraped off. Finally, the LF shell was obtained by drying at room temperature, in a ventilated environment, and away from light. Subsequently, CS-APBA solution (40 mg / mL, grafting degree 15.8%) and polyvinyl alcohol solution (100 mg / mL) were mixed thoroughly at a volume ratio of 1:3 to obtain CP solution. The CP solution was added to the grooves of a PDMS mold containing the shell, and the mold was centrifuged to ensure the CP solution fully filled the needle tip cavity. After centrifugation, more CP solution was added to form a microneedle backing layer. Finally, the microneedles were dried at room temperature in a ventilated, dark place, and then demolded to obtain LCP MN.

[0048] Comparative Example 3 This comparative example provides a silver nanoparticle-loaded microneedle (LAg@CP MN), which is prepared by the following method: 1 g of CS was dissolved in 100 mL of MES buffer solution at pH=5, and stirred for 30 min to ensure complete dissolution. 0.12 g of EDC and 0.072 g of NHS were added, and the mixture was stirred in an ice-water bath for 1 h to activate the carboxyl groups on the CS. Then, 0.107 g of APBA was added, and the reaction proceeded for 24 h. Finally, the solution was dialyzed against deionized water for 3 consecutive days, and then lyophilized for 5 days to obtain CS-APBA.

[0049] An LF-Ag2O2 composite nanoparticle solution (100 mg / mL) was added to the surface of a PDMS mold. Vacuum was then applied to remove air bubbles, ensuring the solution filled the entire needle tip cavity. Excess liquid was then scraped off. Finally, the LF-Ag2O2 shell was obtained by drying at room temperature, in a ventilated environment, and away from light. Subsequently, CS-APBA solution (40 mg / mL, grafting degree 15.8%) and polyvinyl alcohol solution (100 mg / mL) were mixed thoroughly at a volume ratio of 1:3 to obtain CP solution. The CP solution was added to the grooves of a PDMS mold containing the shell, and the mold was centrifuged to ensure the CP solution fully filled the needle tip cavity. After centrifugation, more CP solution was added to form a microneedle backing layer. Finally, the microneedles were dried at room temperature in a ventilated, dark place, and then demolded to obtain LAg@CP MN.

[0050] Experimental Example 1 The morphology and distribution of the LF-Ag2O2 composite nanoparticles prepared in step (1) of Example 2 were observed using transmission electron microscopy. The particle size and distribution of LF-Ag2O2NPs were determined using a nanoparticle size analyzer. Phase analysis of Ag2O, Ag2O2, and LF-Ag2O2NPs was performed using X-ray diffraction (XRD). The ability of LF-Ag2O2NPs to decompose and generate H2O2 in a weakly acidic environment was evaluated by the 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric method.

[0051] The results are as follows Figure 1 As shown. Figure 1 The results in a show that LF-Ag2O2NPs exhibit small particle size and good dispersibility. Figure 1 Figure b shows that the particle size of LF-Ag2O2NPs is approximately 11.7 nm and the distribution is uniform. Figure 1 As shown in Figure c, the characteristic diffraction peaks of LF-Ag2O2NPs (38.13°, 44.31°, 64.46°, 77.42° and 81.57°) correspond one-to-one with Ag2O2 and are significantly different from the characteristic peaks of Ag2O, indicating the successful synthesis of Ag2O2 on LF. Figure 1 The results show that under acidic conditions (pH=4), Ag₂O₂, LF-Ag₂O₂, and H₂O₂ can all convert colorless reduced TMB into blue oxidized TMB, with a distinct absorption peak at 652 nm. This result demonstrates that LF-Ag₂O₂NPs can decompose in a weakly acidic environment, producing silver ions and H₂O₂.

[0052] The morphology of the core-shell microneedles (LAg&FGF@CP MN) prepared in step (2) of Example 2 was characterized by scanning electron microscopy (SEM). In addition, the shell layer was labeled with Rhodamine B (red fluorescence) and the core layer was labeled with fluorescein (green fluorescence), and the core-shell structure of the microneedles was characterized by inverted fluorescence microscopy.

[0053] The results are as follows Figure 2 As shown in a and b. Figure 2 The image in image a shows that the microneedle tips have regular morphology and are evenly arranged. Figure 2 In the image (b), a complete red shell can be seen enclosing a green core, and the red and green fluorescence are clearly separated in space with distinct boundaries, confirming the successful preparation of the microneedle core-shell structure.

[0054] The mechanical strength of the CP MN and LCP MN prepared in Comparative Examples 1 and 2 was characterized using a universal tensile testing machine. The microneedles were cut into microneedle arrays containing 25 needle tips (5×5), and the compression rate was 0.5 mm / min. The load-displacement curves were recorded.

[0055] The results are as follows Figure 2 As shown in Figure c, the load-displacement curves show that the strength of a single-layer CP MN is 0.78 N / needle, while it increases to 0.98 N / needle after adding the LF shell, which far exceeds the force required to penetrate the skin, indicating that it is sufficient to penetrate the skin and biomembranes.

[0056] Experimental Example 2 To investigate the drug release behavior of the inner layer of core-shell microneedles, BSA was added as a model drug to simulate the release of bFGF. The microneedles were prepared according to Example 2, except that bFGF was replaced with BSA at a final concentration of 5 mg / mL. Release curves of the microneedles were tested in PBS solution and PBS solution containing 0.1 mM H2O2.

[0057] The results are as follows Figure 2 As shown in Figure d, the drug release curves reveal that in PBS solution, microneedle release is relatively slow, with a cumulative release rate of 74.28 ± 0.76%. However, in PBS solution containing H2O2, the release behavior of microneedles is significantly accelerated, achieving rapid release of BSA within 4 h, with a cumulative release rate reaching 88.73 ± 1.01% at 48 h. These results indicate that microneedles can rapidly dissociate and release drugs in response to the ROS environment, and their release characteristics match the microenvironment of diabetic wounds.

[0058] Experimental Example 3 Will S. aureus , E. coli MRSA C. albicans Four types of bacteria were co-incubated with the LF-Ag2O2 composite nanoparticles prepared in step (1) of Example 2, and the in vitro antibacterial properties of different concentrations of LF-Ag2O2NPs were evaluated by dilution plate coating method.

[0059] The results are as follows Figure 3 As shown, with the increase of LF-Ag2O2NPs concentration, the number of bacterial colonies gradually decreased, and the bacterial survival rate continued to decline. When the concentration was increased to 6 μg / mL, except for Candida albicans (survival rate 18.38 ± 0.37%), the other three strains showed no colony growth, and the sterilization rate was 100%. This indicates that the LF-Ag2O2 nanoparticles prepared in this invention have excellent antibacterial effects against a variety of strains, including Gram-negative bacteria, Gram-positive bacteria, and fungi, and have broad-spectrum antibacterial activity.

[0060] Furthermore, S. aureus , E. coli MRSA C. albicansFour types of bacteria were co-incubated with CP MN (Comparative Example 1), LCP MN (Comparative Example 2), LAg@CP MN (Comparative Example 3), and LAg&FGF@CP MN (Step 2) of Example 2. Specifically, the microneedle patches containing different components were placed in 0.9 mL of liquid culture medium and mixed with 100 µL of bacterial solution, incubated at 37°C. o The microneedles were cultured in a C-type biochemical incubator for 12 hours. The in vitro antibacterial properties of the four microneedles were then evaluated using the dilution plate coating method.

[0061] The results are as follows Figure 4 As shown. Figure 4 The results showed that there was no significant difference in bacterial colony count between the CP MN treatment group and the blank control group. S. aureus , E. coli MRSA C. albicans The survival rates were 93.78 ± 1.98%, 92.48 ± 1.26%, 96.77 ± 1.58%, and 96.21 ± 2.37%, respectively, indicating that the inner layer microneedles alone do not possess antibacterial ability. The number of colonies decreased slightly after LCP MN treatment. S. aureus , E. coli MRSA C. albicans The survival rates were 79 ± 2.53%, 76.73 ± 2.31%, 92.44 ± 2.35%, and 90.16 ± 1.65%, respectively, indicating that the LF shell endowed the microneedles with a certain antibacterial effect. The number of colonies was significantly reduced after treatment with LAg@CP MN and LAg&FGF@CP MN, with LAg@CP MN treatment showing the largest decrease. S. aureus , E. coli MRSA C. albicans The survival rates were 1.28 ± 0.19%, 0%, 13.08 ± 0.96%, and 27.66 ± 2.01%, respectively, after LAg&FGF@CP MN treatment. S. aureus , E. coli MRSA C. albicans The survival rates were 1.69 ± 0.3%, 0%, 11.5 ± 0.62%, and 21.19 ± 2.99%, respectively. Figure 4 (bc). The results showed that LF-Ag2O2NPs were the main antibacterial components of LAg&FGF@CP MN, and LF and Ag2O2 exerted a synergistic antibacterial effect.

[0062] Test Example 4 The cytotoxicity of CP MN, LCP MN, LAg@CP MN, and LAg&FGF@CP MN prepared in this invention was tested: L929 fibroblasts were seeded at a density of 5000 cells per well in 96-well plates and cultured in a cell culture incubator for 12 h. Subsequently, the cells were co-incubated with extracts of different microneedles for 24 h and 48 h. Cell viability was calculated using the CCK-8 assay after incubation. Furthermore, the biocompatibility of the microneedles was further evaluated using an AO / PI cell viability assay kit, and cell staining was observed under an inverted fluorescence microscope.

[0063] The results are as follows Figure 5 As shown. Figure 5 As shown in Figure a, the green fluorescence intensity of the CP MN, LCP MN, LAg@CP MN and LAg&FGF@CP MN groups was comparable to that of the control group, and there was almost no red fluorescence, indicating that the microneedles have excellent biocompatibility. Figure 5 The results showed that after 24 h and 48 h of treatment with four types of microneedles, the survival rate of L929 cells was higher than 85%, confirming that the microneedles had no obvious cytotoxicity.

[0064] Experimental Example 5 The cell migration-promoting abilities of CP MN, LAg@CP MN, and LAg&FGF@CP MN prepared in this invention were tested: L929 fibroblasts were 10 per well 5 Cells were seeded at a density of 10 μL in 24-well plates and cultured in a cell culture incubator. When the cells reached 90% confluence, the cultured cells were vertically scraped using a 10 μL pipette tip, and floating cells were washed away with PBS. 500 μL of serum-free medium containing different microneedle patches was added, and the cells were cultured together in the cell culture incubator. After 24 h, cell migration images were captured using an inverted fluorescence microscope, and the migration area was quantified and the migration rate calculated using ImageJ software.

[0065] The results are as follows Figure 6 As shown. Figure 6 Figures a and b show that after 24 h, the CP MN group had a limited effect on promoting cell migration, while the cell migration rate of the LAg@CP MN treatment group increased slightly (29.89 ± 3.44%). In contrast, the cell migration rate was significantly increased after the addition of bFGF (89.32 ± 4.01%). This result indicates that the synergistic delivery of LF and bFGF can effectively enhance the performance of microneedles in promoting cell migration.

[0066] Experimental Example 6 To evaluate the promoting effect of LAg&FGF@CP MN prepared in this invention on tissue regeneration in diabetic infected wounds, 6-8 week old female BALB / c mice were selected. To induce type 1 diabetes, mice were fasted for 12 hours and then intraperitoneally injected with streptozotocin (STZ, 150 mg / kg). One week after STZ injection, a random blood glucose level ≥16.7 mmol / L was considered a successful model. STZ-induced diabetic mice were randomly divided into 4 groups of 5 mice each (n=5). The diabetic mice were anesthetized with isoflurane, and their dorsal hair was removed, creating an 8 mm full-thickness circular skin wound on their backs. Each wound was infected with 20 μL of MRSA bacterial suspension (10... 9 (CFU / mL), and then bandaged with 3M bandage. A diabetic mouse MRSA infection wound model was successfully established 2 days after infection. During treatment, the microneedles were cut into 1×1 cm pieces. 2 The microneedles were applied to the wound and secured with a breathable bandage. The microneedles were changed daily for three days. Wound healing and mouse weight changes were recorded on days 0, 3, 7, 10, and 14. Wound area was quantified using ImageJ software, and relative wound area was calculated. Additionally, wound exudate was collected from each group one day after treatment, and wound infection was assessed using the dilution plating method.

[0067] The results are as follows Figure 7 As shown. Figure 7 The results showed that, throughout the treatment period, the LAg&FGF@CP MN group exhibited the fastest healing rate compared to other groups. Figure 7 The results showed that after 3 days of treatment, the relative wound area in the LAg&FGF@CP MN group decreased to 56.66 ± 4.94%, significantly better than that in the control group (80.13 ± 6.88%). On day 7, the wound area in this group was only 14 ± 2.82%, significantly lower than that in the control group. On day 14, the control group and the CP MN group still had wound areas of 11.71 ± 5.02% and 10.96 ± 2.50%, respectively, while the wounds in the LAg&FGF@CP MN group had been almost completely epithelialized. Throughout the treatment period, the body weight of mice in each group did not change significantly. Figure 7 (d).

[0068] Images of bacterial colonies collected from the exudate around the wounds in each group are shown below. Figure 7 In Figure b, a large number of bacterial colonies remained in the control group and the CP MN group, while the colony counts in the LAg@CP MN and LAg&FGF@CP MN groups were significantly reduced, confirming their significant antibacterial ability. This result is consistent with the trend of wound healing, further indicating a close relationship between effective antibacterial activity and promotion of wound healing.

[0069] Experimental Example 7 To further assess wound inflammation and angiogenesis, immunofluorescence staining was performed on CD86, CD206, CD34, and α-SMA in mouse skin tissue 14 days after treatment. The staining procedure for CD86 was as follows: sections were dewaxed, subjected to high-temperature antigen retrieval in sodium citrate buffer (100℃, 3 min), cooled, and washed three times with PBS. The sections were then blocked using Triton-X and BSA in a shaker at 37℃ for 2 hours. Next, CD86 primary antibody (1:400 dilution) was added to the sections, and the sections were incubated in a shaker at 37℃ for 2 hours, followed by two PBS soaks. Then, secondary antibody (1:400 dilution) was added, and the sections were incubated at room temperature in the dark for 1 hour, followed by two PBS soaks. Finally, the sections were mounted with DAPI and observed under an inverted fluorescence microscope. The staining procedures for CD206, CD34, and α-SMA were the same. Finally, the immunofluorescence staining results were quantitatively analyzed using ImageJ software.

[0070] The results of CD86 and CD206 immunofluorescence staining of tissue sections 14 days after treatment are as follows: Figure 8 As shown, the control and CPMN groups exhibited high CD86 expression, indicating a higher number of M1 macrophages in the tissues. In contrast, CD206 expression was significantly increased in the LAg@CP MN and LAg&FGF@CP MN treatment groups. This resulted in an increase in the number of M2 macrophages in the immune microenvironment. This result confirms that LAg&FGF@CP MN can effectively promote the polarization of the M2 macrophage phenotype, remodel the immune microenvironment of the wound, and promote the healing process.

[0071] Immunofluorescence staining was performed on CD34 and α-SMA in tissue sections 14 days after treatment. Figure 9 As shown, CD34 and α-SMA expression was weak in the control group and the CP MN group, while the expression levels of both were significantly enhanced in the LAg@CP MN and LAg&FGF@CP MN groups. This change is attributed to the effective antibacterial effect of LF-Ag2O2 nanoparticles in the early stage, which promotes the orderly transition of the healing process from the inflammatory phase to the proliferative phase. On this basis, the continuous release of bFGF further directly promotes cell proliferation and angiogenesis.

[0072] In summary, the silver-loaded nanoparticles and growth factor core-shell microneedles prepared using the preparation method provided by this invention break the vicious cycle of "infection-inflammation" in infected wounds. By programmatically regulating the wound microenvironment, they significantly improve wound healing efficiency, providing a programmatic treatment strategy for infected skin wounds.

[0073] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A core-shell microneedle loaded with silver nanoparticles and growth factors, characterized in that, The core-shell microneedles include a backing layer and a needle tip array disposed on the backing layer; the needle tips in the needle tip array have a core-shell structure, the shell layer of the needle tips is loaded with protein-Ag2O2 composite nanoparticles, and the core layer of the needle tips is loaded with growth factors. The preparation method of the protein-Ag2O2 composite nanoparticles includes the following steps: The protein aqueous solution was mixed with silver nitrate solution, and then reacted with hydrogen peroxide solution under alkaline conditions to obtain the protein-Ag2O2 composite nanoparticles.

2. The core-shell microneedles of silver-loaded nanoparticles and growth factors according to claim 1, characterized in that, The concentration of the protein aqueous solution is 20 mg / mL, the concentration of the silver nitrate solution is 0.1 M, and the mass fraction of the hydrogen peroxide solution is 30%. The volume ratio of the protein aqueous solution, the silver nitrate solution, and the hydrogen peroxide solution is 5:(0.0625-0.5):(0.0065-0.052).

3. The core-shell microneedles of silver-loaded nanoparticles and growth factors according to claim 1, characterized in that, The pH of the alkaline conditions is 10-12; The mixing reaction was carried out at a temperature of 25 ± 3℃ for 20-60 min.

4. The core-shell microneedles of silver-loaded nanoparticles and growth factors according to any one of claims 1-3, characterized in that, The proteins include lactoferrin, serum albumin, transferrin, immunoglobulins, and hemoglobin.

5. The core-shell microneedles of silver-loaded nanoparticles and growth factors according to any one of claims 1-3, characterized in that, The growth factors include recombinant fibroblast growth factor.

6. A method for preparing core-shell microneedles of silver-loaded nanoparticles and growth factors according to any one of claims 1-5, characterized in that, Includes the following steps: The protein-Ag2O2 composite nanoparticles were injected into a mold to form a needle-tip shell; The growth factor is mixed with the matrix material and injected into the cavity of the needle tip shell to form the needle tip core layer; The matrix material is used to form a backing layer to obtain the core-shell microneedles.

7. The preparation method according to claim 6, characterized in that, The matrix material is formed by cross-linking chondroitin sulfate grafted with 3-aminophenylboronic acid with polyvinyl alcohol; The concentration of chondroitin sulfate grafted with 3-aminophenylboronic acid is 35-45 mg / mL; the concentration of polyvinyl alcohol is 90-120 mg / mL; and the volume ratio of chondroitin sulfate grafted with 3-aminophenylboronic acid to polyvinyl alcohol is 1:1-1:

5.

8. The preparation method according to claim 6, characterized in that, After the growth factor is mixed with the matrix material, the final concentration of the growth factor is 5-20 ng / mL.

9. The use of the core-shell microneedles of silver-loaded nanoparticles and growth factors as described in any one of claims 1-5 in the preparation of a medicament for treating infected skin wounds.

10. A medicine for treating infected skin wounds, characterized in that, The drug comprises core-shell microneedles containing silver-loaded nanoparticles and growth factors as described in any one of claims 1-5.

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