Multifunctional mixed microneedle based on efficient delivery of NMN-coated Cu / CeO2 nano enzyme and preparation method and application thereof

Abstract

The invention discloses a multifunctional mixed microneedle based on efficient delivery of NMN-coated Cu / CeO2 nano-enzyme as well as a preparation method and application of the multifunctional mixed microneedle, and belongs to the technical field of biomedical materials. The preparation method of the multifunctional mixed microneedle comprises the following steps: under a water bath heating condition, adding freeze-dried methacryloyl gelatin, NMN-coated Cu / CeO2 nano-enzyme and a photoinitiator into deionized water to obtain a microneedle tip precursor solution; adding polyvinyl alcohol and phenylboronic acid grafted polylysine into deionized water to obtain a microneedle substrate precursor solution; and adding the microneedle tip precursor solution into a microneedle mold for vacuum defoaming concentration treatment, then carrying out blue light curing crosslinking, then adding the microneedle substrate precursor solution into the microneedle mold, and then carrying out drying demolding to prepare the multifunctional mixed microneedle based on efficient delivery of the NMN-coated Cu / CeO2 nano-enzyme. The drug-loaded nano-enzyme in the microneedle has high SOD and CAT enzyme activity, oxidative stress in the wound surface can be solved, the delivered drug NMN can improve the immune microenvironment and recover the mitochondrial function, meanwhile, the drug-loaded nano-enzyme is efficiently delivered to the wound surface by using the microneedle patch, and the problems of infection and bleeding of the diabetic wound surface can be solved at the same time.

Description

Technical Field

[0001] This invention belongs to the field of biomedical materials technology, specifically relating to a multifunctional hybrid microneedle based on efficient delivery of NMN@Cu / CeO2 nanozymes, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus, a systemic metabolic disease, has become a widespread public health problem that seriously threatens human health. Characterized by hyperglycemia, diabetes can lead to various health complications, including diabetic foot (DFU), cardiovascular disease, neurological disorders, and chronic renal failure, severely endangering human health. The pathogenesis of diabetic wounds is complex, involving bacterial infection, oxidative stress imbalance, increased inflammatory cytokines, and nicotinamide adenine dinucleotide (NAD). + Diabetic wounds exhibit complex characteristics such as impaired biosynthesis, reduced nerve and angiogenesis. Oxidative stress is considered one of the main culprits contributing to the difficulty in healing diabetic wounds. In a prolonged high-glucose environment, excessive glucose load triggers increased ROS production in mitochondria, leading to mitochondrial dysfunction and antioxidant system imbalance, subsequently causing cell dysfunction and death. Furthermore, persistent chronic inflammation in the wound can also cause local neurological and microvascular lesions, disrupt the immune microenvironment, exacerbate wound hypoxia and ischemia, and further hinder wound healing. Therefore, comprehensively addressing these key issues may be a new direction for future treatment of diabetic wounds.

[0003] Studies have reported the presence of NAD+ in patients with diabetes mellitus (DM). + The decline in levels can be addressed by supplementing NAD. + or NAD + As a precursor, it can significantly improve mitochondrial dysfunction, maintain redox homeostasis, and promote the recovery of immune cell function, which is crucial for promoting wound healing in diabetic patients. Nicotinamide mononucleotide (NMN) is an NAD+ precursor. + The direct precursor of NMN can be converted into NAD+ via the NR / NRK pathway to achieve its biological functions. Researchers have demonstrated that NMN supplementation can enhance NAD+. +Levels of NMN supplementation can improve wound healing and blood reperfusion in diabetic mice after ischemia. Supplementation with NMN increased Sirtuin1 (SIRT1) activity and reversed vascular dysfunction and oxidative stress in mice with age-related diseases. NMN supplementation plays a crucial role in regulating mitochondrial swelling, glucose metabolism, maintaining energy metabolism, preventing oxidative stress, and restoring mitochondrial homeostasis. However, due to the short half-life of NMN, achieving maximum therapeutic efficacy through wound delivery is challenging. Furthermore, bacterial infection of wounds caused by high-glucose environments is a significant concern, and prolonged infection can exacerbate wound healing problems. Because of their excellent physicochemical properties, nanoparticles offer a novel approach to treating diabetic wounds by developing a novel nanoenzyme with "dual" antioxidant activity to deliver NMN to wounds, synergistically combating bacteria, reducing oxidative stress, promoting angiogenesis, and improving the immune microenvironment.

[0004] However, direct application of nanozymes to diabetic wounds has limitations such as uneven distribution, difficulty in preservation, and potential biotoxicity. Therefore, this study focuses on modifying nanozymes to enable them to carry NMN drugs while addressing the issues of uneven nanoscale distribution and biotoxicity. Furthermore, the aim is to design a wound dressing that can efficiently deliver drug carriers to the wound, addressing both infection and bleeding issues in diabetic wounds. Improving wound angiogenesis and revascularization in diabetic patients is crucial for managing chronic diabetic wounds and accelerating wound healing. Summary of the Invention

[0005] To overcome the shortcomings of the prior art, the present invention aims to provide a multifunctional hybrid microneedle based on efficient delivery of NMN@Cu / CeO2 nanozymes, its preparation method and application. The high SOD and CAT enzyme activity of the drug-loaded nanozyme itself can solve the oxidative stress in the wound, and the delivery of NMN can improve the immune microenvironment and restore mitochondrial function. At the same time, the use of microneedle patches to efficiently deliver the drug-loaded nanozyme to the wound can simultaneously solve the problems of infection and bleeding in diabetic wounds.

[0006] To achieve the above objectives, the present invention employs the following technical solution: This invention provides a method for preparing a multifunctional hybrid microneedle based on a highly efficient NMN@Cu / CeO2 nanozyme delivery system, comprising the following steps: Under water bath heating conditions, freeze-dried methacryloyl gelatin was added to deionized water to prepare a precursor solution. NMN@Cu / CeO2 nanozyme and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate were mixed with the precursor solution to obtain microneedle tip precursor solution. Under water bath heating conditions, polyvinyl alcohol and polylysine grafted with phenylboronic acid were added to deionized water to obtain a microneedle substrate precursor solution. The microneedle tip precursor solution was added to the microneedle mold for vacuum defoaming and concentration, then blue light curing and crosslinking were performed. The microneedle base precursor solution was then added to the microneedle mold and dried and demolded to obtain a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes.

[0007] In one embodiment, the mass ratio of lyophilized methacryloyl gelatin, NMN@Cu / CeO2 nanozyme, and deionized water in the precursor solution is (50~150):(0.08~0.12):1; the concentration of NMN@Cu / CeO2 nanozyme in the microneedle tip precursor solution is 80~120 μg / mL; and the concentration of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite in the microneedle tip precursor solution is 0.25%.

[0008] In one embodiment, the concentration of polyvinyl alcohol in the microneedle substrate precursor solution is 200 mg / mL, and the concentration of phenylboronic acid-grafted polylysine is 50-120 mg / mL.

[0009] In one embodiment, the preparation method of the NMN@Cu / CeO2 nanozyme is as follows: Cu / CeO2 nanoparticles were suspended in a first NaOH solution and stirred. Then, C3H5Cl and a second NaOH solution were added sequentially and stirred overnight at room temperature to obtain the first reaction product. The first reaction product was centrifuged and washed until the first supernatant of the first reaction product was neutral. Then, the first supernatant was vacuum dried to obtain the dried product. The dried product and polyethyleneimine (PEI) were dissolved in deionized water and stirred to obtain the second reaction product. The second reaction product was centrifuged and washed until the second supernatant of the second reaction product was neutral. Then, it was dialyzed and centrifuged in sequence to remove the supernatant and then freeze-dried to obtain PEI-modified Cu / CeO2. The PEI-modified Cu / CeO2 and NMN aqueous solution were then mixed and incubated at room temperature with stirring. After centrifugation and washing to remove the supernatant, NMN@Cu / CeO2 nanozyme was prepared and resuspended in deionized water for storage.

[0010] In one embodiment, the concentration of the first NaOH solution is 0.1M; the concentration of the second NaOH solution is 2M; and the ratio of the Cu / CeO2 nanoparticles, the first NaOH solution, C3H5Cl, and the second NaOH solution is 50mg:10mL:10mL:0.5mL. The concentration of polyethyleneimine (PEI) is 0.004 M; the ratio of the dried product, PEI, and deionized water is 50 mg: 40 mg: 250 mL. The concentration of the NMN aqueous solution is 0.0075M; the ratio of the amount of PEI-modified Cu / CeO2 and NMN aqueous solution is 50mg:1mL.

[0011] In one embodiment, the method for preparing the phenylboronic acid-grafted polylysine is as follows: 2-(N-morpholino)ethanesulfonic acid (MES) was dissolved in deionized water and mixed thoroughly until the solution was clear and transparent. Then, polylysine (EPL), phenylboronic acid (PBA), N,N-dimethylformamide (DMF), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were added to the solution in sequence, and the mixture was stirred overnight at 60°C. After the solution returned to room temperature, the supernatant was collected by centrifugation. The supernatant was dialyzed with deionized water, and finally, the dialyzed solution was freeze-dried to obtain phenylboronic acid-grafted polylysine.

[0012] In one embodiment, the ratio of 2-(N-morpholino)ethanesulfonic acid (MES), deionized water, polylysine (EPL), phenylboronic acid (PBA), N,N-dimethylformamide (DMF), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) is 2g:200mL:10g:2.64g:20mL:6.1g:3.66g.

[0013] In one embodiment, the water bath heating temperature is 50~60℃; the vacuum defoaming and concentration treatment is performed 2~3 times; the blue light curing and crosslinking time is 30~60s; the drying time is 24~72h, and the drying temperature is 30~35℃.

[0014] The present invention also provides a multifunctional hybrid microneedle based on the highly efficient delivery of NMN@Cu / CeO2 nanozyme, prepared by the above-described method for preparing a multifunctional hybrid microneedle based on the highly efficient delivery of NMN@Cu / CeO2 nanozyme.

[0015] In another aspect, the present invention provides the application of the multifunctional hybrid microneedles based on the highly efficient delivery of NMN@Cu / CeO2 nanozymes, prepared by the above-described method, in the preparation of wound dressings for the treatment of diabetes.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for preparing a multifunctional hybrid microneedle based on a highly efficient NMN@Cu / CeO2 nanozyme delivery system. The microneedle precursor solution comprises two components: a microneedle base precursor solution and a microneedle tip precursor solution. The microneedle base precursor solution contains PVA and EPL-PBA. Polylysine exerts cationic antibacterial activity, seals wounds, stops bleeding (cations react with the negative charges on platelets to aggregate platelets), and promotes wound repair. The hydroxyl groups of the PVA base coordinate with the carboxyl groups of tissue proteins to enhance adhesion. The phenylboronic acid groups of EPL-PBA crosslink with the hydroxyl groups of PVA to form dynamic borate ester bonds with pH and ROS responsiveness, which then crosslink into a gel, forming a polymer network. When applied to wounds, in the high ROS environment of the wound, the ROS-responsive borate ester bonds break, releasing EPL with bactericidal function for responsive bactericidal action. The microneedle tip precursor solution comprises GelMA and NMN@Cu / CeO2 nanozymes. The methacryloyl gelatin component exhibits excellent biocompatibility, degradability, and non-immunogenicity, while also promoting extracellular matrix (ECM) formation, facilitating cross-linking under physiological conditions, and being easy to prepare and enzymatically degraded, demonstrating its potential in cartilage tissue engineering. Furthermore, the microneedle tip possesses sufficient mechanical strength to deliver the nanozymes deep into the tissue to exert their effects.

[0017] Furthermore, in the preparation of the microneedle tip, lyophilized methacryloyl gelatin, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and NMN@Cu / CeO2 nanozyme were dissolved in deionized water to obtain the microneedle tip precursor solution. Firstly, methacryloyl gelatin (GelMA), prepared based on gelatin (a type of deformed collagen), was specifically chosen as the main component of the microneedle tip due to its excellent biocompatibility, degradability, and non-immunogenicity, making it a promising candidate for manufacturing wound dressings. Utilizing the tip to deliver nanozyme into deep tissues enhances the antibacterial effect of the microneedle; the released NMN and Cu / CeO2 synergistically enhance ROS clearance in the tissue and promote wound angiogenesis. By promoting tissue adhesion, hemostasis, bacterial elimination, ROS removal, and significantly promoting the migration, proliferation, and angiogenesis of human umbilical vein endothelial cells (HUVECs), GelMA creates a protective environment for diabetic wound healing. Furthermore, by promoting angiogenesis, reducing inflammation, and increasing the number of healing-promoting M2 macrophages, it avoids the high-cost and time-consuming processes required for growth factor or cell / cell-derived therapies. In addition to these advantages, GelMA also promotes extracellular matrix (ECM) formation, is easily cross-linked under physiological conditions, is easy to prepare, and is readily enzymatically degradable, demonstrating its potential in cartilage tissue engineering. Moreover, needles containing GelMA can absorb a certain amount of water and interstitial fluid after drying, causing them to swell and soften. This helps them adapt to irregular wounds, increases their adhesion, and provides a physical barrier, keeping bacteria out and water in, thus achieving hemostasis through physical closure. Most importantly, the dried tips of microneedles containing GelMA have a certain mechanical strength, allowing them to penetrate the stratum corneum of the skin and efficiently deliver NMN@Cu / CeO2 nanoenzymes to deep tissues, achieving precise drug release.

[0018] Furthermore, in the preparation of the microneedle substrate, a precursor solution for the microneedle substrate is prepared by mixing polyvinyl alcohol (PVA) with phenylboronic acid-grafted polylysine. Phenolic acid-grafted polylysine (EPL-PBA) is then prepared through an amidation reaction between the carboxyl groups in phenylboronic acid and the amino groups in polylysine. Subsequently, the phenylboronic acid bonds (EPL-PBA) in the phenylboronic acid-grafted polylysine react with the hydroxyl groups in PVA to form dynamic borate ester bonds. Secondly, the aldehyde C=O double bonds in the precursor solution can react with the amino group (NH2) through a Schiff base reaction to generate C=N double bonds, crosslinking to form a gel. The dynamic borate ester bonds formed in the microneedle substrate exhibit certain ROS and pH-responsive functions.

[0019] In summary, the microneedles described in this invention possess high affinity for amino groups and papery peptides based on the catechol groups of PVA, EPL-PBA, and GelMA, forming strong covalent bonds, π-π interactions, and hydrogen bonds with various substances. These principles explain the tissue adhesion mechanism of the microneedle patch. Simultaneously, the positively charged amino groups in polylysine interact electrostatically with the negatively charged carboxyl groups in tissue, further enhancing the bioadhesion strength of the microneedles. This broad adhesion to target tissues provides the microneedles with the potential for extensive practical medical applications in wound healing.

[0020] The multifunctional hybrid microneedles based on the efficient delivery of NMN@Cu / CeO2 nanozymes provided by this invention represent a promising wound-closing hemostatic barrier. This is because they possess the natural hemostatic properties of GelMA, positively charged polylysine, and excellent adhesion, thereby synergistically enhancing hemostatic activity. The inherent regenerative activity of NMN, along with the synergistic effect of Cu / CeO2, accelerates the healing time of diabetic wounds. Furthermore, the moist environment provided by the absorbent interstitial fluid microneedles also promotes wound healing. As a dressing, the microneedles can integrate multiple active molecules with different functions to promote the healing of diabetic wounds.

[0021] This invention provides a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes, which can be used to prepare wound dressings for treating diabetes. Gel microneedles (MNs) that can bypass physical barriers (such as the stratum corneum) and directly deliver the effective payload to deep tissues have become a novel transdermal drug delivery method, showing extremely broad application prospects. Compared with traditional wound dressings, gel microneedles have a series of advantages such as high drug permeability, large drug loading capacity, and rapid onset of action. Based on these excellent properties, MNs have been widely used in various biomedical fields, including scalp regeneration, diabetic wounds, hypertrophic scars, and cardiac repair. The aforementioned hybrid microneedle uses NMN@Cu / CeO2 nanozymes as a dressing repair system to promote the healing of diabetic wounds. The simultaneous introduction of NMN and Cu / CeO2 molecules into the dressing is beneficial for skin wound repair. Simultaneously, microneedles possess the ability to regulate and precisely release therapeutic substances and promote wound healing by simultaneously loading a large number of bioactive compounds into the hydrogel network. This enables long-term release and improves cellular behavior. During microneedle fabrication, NMN and Cu / CeO2 are encapsulated in the needle tip, giving the microneedles excellent ROS scavenging capabilities, which aids wound healing. NMN@Cu / CeO2-MN has the potential to reduce excessive oxidative stress and protect cells in the microenvironment of diabetic wounds. Furthermore, the synergistic antibacterial effect of EPL and Cu / CeO2 is the reason for the improved antibacterial efficiency. The strong electrostatic interaction between the positively charged EPL and the negatively charged bacterial phospholipid membrane components leads to bacterial death through membrane decomposition. 2+There are three potential mechanisms limiting microbial growth: First, metal ions can directly disrupt ATP synthesis and DNA replication by crossing the cell membrane; second, Cu... 2+ It may use electrostatic forces to fix the cell membrane, impairing cell integrity and preventing protons and other molecules from effectively passing through the cell membrane; finally, metal ions induce oxidative stress by generating ROS, which can disrupt the normal activity of bacteria and ultimately kill bacteria by destroying the bacterial membrane, DNA and mitochondria. The combination of all these properties significantly enhances the antibacterial activity of the hydrogel. Attached Figure Description

[0022] Figure 1 These are macroscopic and SEM scan images of the microneedle patch of the present invention; Figure 2 These are 3D fluorescence images of the microneedles of the present invention after different fluorescent staining. Figure 3 This is a statistical chart showing the microneedle swelling performance test results of the present invention; Figure 4 This is the FT-IR spectrum of the microneedle patch of the present invention; Figure 5 Liquid nuclear magnetic resonance (NMR) 1H spectrometry analysis of the microneedle substrate and tip (EPL-PBA and GelMA) of this invention; Figure 6 This is a schematic diagram showing the in vitro rhodamine release (μg / mL) of the NMN@Cu / CeO2-MN microneedles of the present invention; Figure 7 A representative schematic diagram of live and dead cell staining after HUVECs were co-cultured with MN, Cu / CeO2-MN, NMN-MN and NMN@Cu / CeO2-MN of the present invention for 24 h. Figure 8 A representative schematic diagram of live and dead cell staining after HFB was co-cultured with MN, Cu / CeO2-MN, NMN-MN and NMN@Cu / CeO2-MN of the present invention for 24 h. Figure 9 The graph shows the reactive oxygen species (ROS) scavenging ability test results of the MN, Cu / CeO2-MN, NMN-MN and NMN@Cu / CeO2-MN microneedles of the present invention. Figure 10 The images show the antibacterial activity test results of the MN, Cu / CeO2-MN, NMN-MN and NMN@Cu / CeO2-MN microneedles of the present invention; wherein, Figure (A) is the antibacterial activity test image with Staphylococcus aureus, and Figure (B) is the antibacterial activity test image with Escherichia coli. Figure 11Figure 1 shows the hemostatic performance test results of NMN@Cu / CeO2-MN in the tail and liver hemorrhage models of C57 mice according to the present invention; wherein, Figure (A) is a schematic diagram of the mouse incision model; Figure (B) is a representative hemorrhage photograph; and Figure (C) is a statistical chart of the amount of bleeding. Figure 12 The in vivo diabetic wound healing performance test diagram of the MN, Cu / CeO2-MN, NMN-MN and NMN@Cu / CeO2-MN microneedles of the present invention; Figure 13 The image shows statistical data on wound size of the MN, Cu / CeO2-MN, NMN-MN, and NMN@Cu / CeO2-MN microneedles of the present invention and the control group. Figure 14 This is a statistical chart showing the number of CD31-positive neovascularizations in the MN, Cu / CeO2-MN, NMN-MN, and NMN@Cu / CeO2-MN microneedles of the present invention and the control group. Figure 15 This is a schematic diagram illustrating the synthesis process of the multifunctional hybrid microneedles based on the efficient delivery of NMN@Cu / CeO2 nanozymes according to the present invention. Detailed Implementation

[0023] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0024] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0025] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0026] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0027] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0028] To overcome the shortcomings of existing technologies, such as short half-life of NMN, drug leakage, peri-wound swelling, low drug concentration in the wound center, lack of antibacterial effect of NMN, increased risk of infection due to continuous wound exposure, and loss of wound moisture, and inability to further clear ROS and promote angiogenesis, this invention provides a multifunctional hybrid microneedle based on efficient delivery of NMN@Cu / CeO2 nanozymes, its preparation method, and its application. Using microneedle patches as a novel wound dressing, it can achieve efficient delivery of NMN to the wound. The released Cu / CeO2 nanozymes and polylysine provide dual antibacterial effects. Through the multiple functions of NMN@Cu / CeO2-MN microneedles, it can enhance ROS clearance and promote wound angiogenesis.

[0029] like Figure 15 As shown, a method for preparing a multifunctional hybrid microneedle based on efficient delivery of NMN@Cu / CeO2 nanozymes is provided, including the following steps: Under water bath heating conditions, freeze-dried methacryloyl gelatin (GelMA) was added to deionized water to prepare a precursor solution. NMN@Cu / CeO2 nanozyme and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite (LAP) were mixed with the precursor solution to obtain microneedle tip precursor solution. Under water bath heating conditions, polyvinyl alcohol (PVA) and phenylboronic acid-grafted polylysine (EPL-PBA) were added to deionized water to obtain a microneedle substrate precursor solution. The microneedle tip precursor solution was added to the PDMS microneedle mold and concentrated under vacuum negative pressure for 2-3 times, and then cured and crosslinked under blue light. The microneedle substrate precursor solution was added to the PDMS microneedle mold and dried to demold, thus obtaining a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes.

[0030] The preparation method of the lyophilized methacrylamide gelatin (GelMA) used in the above preparation method is as follows: Pigskin gelatin powder was dissolved in PBS (phospho-1 phosphate buffer) and stirred in an oil bath at 60°C until the gelatin was completely dissolved to obtain a solution. The solution was cooled to 50°C and kept at this constant temperature. Methacrylic anhydride (MA) was added to the solution, and the mixture was stirred vigorously. Then, PBS (phosphodiesterase buffer) was added to terminate the reaction. The solution was then dialyzed and then lyophilized at low temperature to recover GelMA.

[0031] The ratio of the above-mentioned pigskin gelatin powder, phosphate-buffered saline (PBS), methacrylic anhydride (MA), and phosphate-buffered saline (PBS) is 20 g: 180 mL: 16 mL: 800 mL.

[0032] The preparation method of the NMN@Cu / CeO2 nanozyme used in the above preparation method is as follows: Cu / CeO2 nanoparticles were suspended in a 0.1M NaOH solution and stirred. Then, C3H5Cl and a 2M NaOH solution were added sequentially and stirred overnight at room temperature to obtain the first reaction product. The ratio of Cu / CeO2 nanoparticles, the first NaOH solution, C3H5Cl, and the second NaOH solution was 50mg:10mL:10mL:0.5mL. The first reaction product was centrifuged and washed until the first supernatant of the first reaction product reached neutrality. Then, the first supernatant was vacuum dried to obtain the dried product (the drying quality was based on Cu / CeO2). The dried product was dissolved in deionized water with polyethyleneimine (PEI) at a concentration of 0.004 M and stirred to obtain the second reaction product; wherein the ratio of dried product, polyethyleneimine (PEI) and deionized water was 50 mg: 40 mg: 250 mL. The second reaction product was centrifuged and washed until the second supernatant of the second reaction product was neutral. Then, it was dialyzed and centrifuged in sequence to remove the supernatant and then freeze-dried to obtain PEI-modified Cu / CeO2. The PEI-modified Cu / CeO2 and a 0.0075M NMN aqueous solution were mixed and incubated at room temperature with stirring. The supernatant was then removed by centrifugation and washing to obtain NMN@Cu / CeO2 nanozyme, which was then resuspended in deionized water for storage. The ratio of PEI-modified Cu / CeO2 to NMN aqueous solution was 50mg:1mL.

[0033] The method for preparing the phenylboronic acid-grafted polylysine is as follows: 2-(N-morpholino)ethanesulfonic acid (MES) was dissolved in deionized water and mixed thoroughly until the solution was clear and transparent. Then, polylysine (EPL), phenylboronic acid (PBA), N,N-dimethylformamide (DMF), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were added to the solution in sequence, and the mixture was stirred overnight at 60°C. After the solution returned to room temperature, the supernatant was collected by centrifugation. The supernatant was dialyzed with deionized water, and finally, the dialyzed solution was freeze-dried to obtain phenylboronic acid-grafted polylysine.

[0034] The ratio of the above-mentioned 2-(N-morpholino)ethanesulfonic acid MES, deionized water, polylysine EPL, phenylboronic acid PBA, N,N-dimethylformamide DMF, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC, and N-hydroxysuccinimide NHS is 2g:200mL:10g:2.64g:20mL:6.1g:3.66g.

[0035] This invention provides a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes, prepared according to the above-described method for preparing a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes.

[0036] The present invention also provides the application of the above-mentioned highly efficient delivery of NMN@Cu / CeO2 nanozymes in the preparation of wound dressings for the treatment of diabetes.

[0037] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0038] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0039] The specific synthesis methods of GelMA, NMN@Cu / CeO2 nanozyme and EPL-PBA used in the embodiments of this invention are as follows: The specific synthesis method of GelMA is as follows: First, 20 g of porcine skin gelatin powder (Sigma-Aldrich) was dissolved in 180 mL of PBS (phosphate-buffered saline) and stirred in an oil bath at 60 °C until the gelatin was completely dissolved. The solution was cooled to 50 °C and maintained at this constant temperature. 16 mL of methacrylic anhydride (MA) was added to the solution, and the mixture was stirred vigorously for 1 h. Subsequently, 800 mL of PBS was added to terminate the reaction. The above solution was dialyzed against deionized water at 40 °C in the dark for 24 h. Finally, the dialyzed solution was lyophilized at -80 °C to recover GelMA for further use.

[0040] The specific synthesis method of NMN@Cu / CeO2 nanozyme is as follows: First, Cu / CeO2 nanoparticles were synthesized. A CeN3O9·6H2O aqueous solution (0.5M, 2ml) was added to 60ml of ethylene glycol, and the mixture was vigorously stirred at room temperature for 40min. The resulting solution was transferred to a high-pressure reactor and kept in an oven at 180℃ for 12h to obtain CeO2 nanoparticles. 57.85mg of Cu(NO3)2 was added to the CeO2 product obtained above, and the mixture was stirred for 1h, then kept in an high-pressure reactor at 180℃ for another 4.5h. The precipitate was collected by centrifugation, washed alternately with anhydrous ethanol and deionized water, and then freeze-dried to obtain hollow copper-doped cerium oxide nanoparticles (abbreviated as Cu / CeO2).

[0041] Next, PEI was used to modify the surface charge of Cu / CeO2 nanoparticles, which can be achieved through the SN2 reaction between PEI and C3H5Cl.

[0042] First, 50 mg of Cu / CeO2 was suspended in 10 mL of 0.1 M NaOH solution and stirred at room temperature for 10 min. Then, 10 mL of C3H5Cl and 0.5 mL of 2 M NaOH were added sequentially, and the mixture was stirred overnight at room temperature. The reaction product was centrifuged at 10,000 rpm (5 min) and washed several times with deionized water until the pH of the supernatant was approximately 7. The product was then dried under vacuum and stored for further use. 50 mg of the dried product and 40 mg of PEI (0.004 M) were dissolved in 250 mL of deionized water and stirred at room temperature for 24 h. The reaction product was washed several times with deionized water at 4,000 rpm (20 min) until the pH of the supernatant was approximately 7. Next, the mixture was dialyzed against deionized water for 24 h using a dialysis membrane (MWCO = 10 kDa) to remove unreacted solvent. Finally, the mixture was centrifuged at 4,000 rpm for 20 min, and the supernatant was discarded. PEI-modified Cu / CeO2 (abbreviated as PEI-Cu / CeO2NPs) were obtained by vacuum freeze-drying at -80℃. To achieve successful loading of NMN in PEI-Cu / CeO2, 50 mg of PEI-Cu / CeO2 and 1 mL of an aqueous solution of NMN (0.0075 M) were mixed and incubated at room temperature with stirring at 100 rpm for 24 h. The solution was then washed twice with deionized water at 10000 rpm (20 min), centrifuged to remove the supernatant, and finally resuspended in deionized water for further use (abbreviated as NMN@Cu / CeO2NPs).

[0043] The specific preparation process of EPL-PBA is as follows: 2g of MES (2-(N-morpholino)ethanesulfonic acid) was dissolved in 200mL of deionized water and mixed thoroughly until the solution was clear and transparent. Then, 10g of EPL (polylysine), 2.64g of PBA (phenylboronic acid), 20mL of LDMF (N,N-dimethylformamide), 6.1g of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), and 3.66g of NHS (N-hydroxysuccinimide) were added sequentially to this solution. The solution was stirred overnight at 60℃. After the solution returned to room temperature, it was centrifuged at 7000rpm for 10min, and the supernatant was collected. The supernatant was dialyzed with deionized water for 24-72 hours. Finally, the dialyzed solution was freeze-dried under vacuum at -80℃ to obtain EPL-PBA for later use.

[0044] The preparation process of NMN@Cu / CeO2-MN is as follows: Step 1: Add methacryloyl gelatin (GelMA) to deionized water under water bath heating conditions at 50~60℃ to obtain a precursor solution; wherein, the water bath heating conditions are preferably 60℃; the concentration of methacryloyl gelatin (GelMA) is preferably 5%, 10% and 15%.

[0045] Specifically, weigh 50-150 mg of lyophilized methacryloyl gelatin (GelMA) and place it in a 5 mL centrifuge tube. Add 1 mL of deionized water and dissolve at 50-60 °C for 1 h. After complete dissolution, obtain the precursor solution.

[0046] Step 2: The NMN@Cu / CeO2 nanozyme, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite, and the precursor solution are mixed to obtain the microneedle tip precursor solution; wherein the concentration of the NMN@Cu / CeO2 nanozyme is 80~120 μg / mL, and the concentration of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite is 0.25%.

[0047] Specifically, add 10-15 μL of NMN@Cu / CeO2 nanozyme (8 mg / mL is the mother solution concentration) to the precursor solution, and mix thoroughly by ultrasonication until homogeneous to obtain the microneedle tip precursor solution.

[0048] Step 3: Add the microneedle tip precursor solution into the PDMS mold, maintain it under vacuum for 5 minutes to remove air bubbles from the tip, and irradiate it with blue light for 30-60 seconds to crosslink it into a gel, thus obtaining the microneedle tip.

[0049] Specifically, the process is as follows: First, the microneedle tip precursor solution is centrifuged to remove bubbles and maintained in a liquid state at 60°C. The microneedle tip precursor solution is then added to a PDMS mold, and the mold is placed under vacuum for 5 minutes to remove air bubbles from the tip. Next, the mold is heated at 50°C for 30 minutes. This process is repeated 2-3 times to obtain concentrated tips. Finally, blue light is used to irradiate the tips for 30-60 seconds to crosslink them into a gel.

[0050] Step 4: Add polyvinyl alcohol (PVA) and phenylboronic acid-grafted polylysine (EPL-PBA) to deionized water under water bath heating conditions of 50~60℃ to obtain microneedle substrate precursor solution; wherein, the concentration of polyvinyl alcohol in microneedle substrate precursor solution is 200mg / mL, and the concentration of phenylboronic acid-grafted polylysine is 50~150mg / mL.

[0051] Specifically, 200 mg of polyvinyl alcohol (PVA) was weighed and placed in a 5 mL centrifuge tube. 1 mL of deionized water was added and dissolved at 50-60 °C for 1 h. Then, 50-150 mg of lyophilized phenylboronic acid-grafted polylysine (EPL-PBA) was added to the solution and thoroughly mixed until homogeneous. After complete dissolution, the microneedle substrate precursor solution was obtained.

[0052] Step 5: Add an appropriate amount of microneedle substrate precursor solution to the PDMS mold, and place the mold in an oven to dry for 24-72 hours. Demold the microneedles to obtain the prepared microneedle patch.

[0053] Specifically, the substrate precursor solution is centrifuged to remove bubbles, then an appropriate amount of precursor solution is added to PDMS, and the mold is placed in an oven to dry. The drying temperature is maintained at 30~35℃, preferably 30℃, and the drying time is 24~72 hours. After drying, the microneedles are demolded to obtain the prepared microneedle patch. This invention provides a novel multifunctional NMN@Cu / CeO2-MN microneedle with biocompatibility, adhesion, antibacterial properties, sustained release of NMN and Cu / CeO2, ROS scavenging, and angiogenesis promotion. This provides a new therapeutic target and approach for the treatment of diabetic wounds and lays the foundation for the clinical application of novel hydrogels. The aforementioned multifunctional NMN@Cu / CeO2-MN microneedle, based on the synergistic effect of NMN and Cu / CeO2, can be used in the preparation of wound dressings for the treatment of diabetes.

[0054] Example 1 This embodiment provides a method for preparing a multifunctional hybrid microneedle based on a highly efficient NMN@Cu / CeO2 nanozyme delivery system, comprising the following steps: Step 1: Add lyophilized methacryloyl gelatin (GelMA) to deionized water heated in a water bath at 60°C to obtain a precursor solution; wherein the concentration of methacryloyl gelatin (GelMA) in the precursor solution is 5%.

[0055] Step 2: The NMN@Cu / CeO2 nanozyme, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite, and the precursor solution are mixed to obtain the microneedle tip precursor solution; wherein, the concentration of NMN@Cu / CeO2 nanozyme in the microneedle tip precursor solution is 100 μg / mL, and the concentration of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite is 0.25%.

[0056] Step 3: Add the microneedle tip precursor solution into the PDMS microneedle mold, maintain it under vacuum for 5 minutes to remove air bubbles from the tip, repeat the defoaming step 2-3 times, and use blue light to irradiate for 30 seconds to crosslink it into a gel, thus obtaining the microneedle tip.

[0057] Step 4: Weigh 200mg of polyvinyl alcohol (PVA) into a 5mL centrifuge tube, add 1mL of deionized water, and dissolve at 50~60℃ for 1h. Then add 50mg of lyophilized phenylboronic acid-grafted polylysine (EPL-PBA) to the solution, mix thoroughly until homogeneous, and obtain the microneedle substrate precursor solution after complete dissolution.

[0058] Step 5: Centrifuge the microneedle substrate precursor solution to remove bubbles, then add an appropriate amount of precursor solution to the PDMS mold, place the mold in an oven to dry, maintain the drying temperature at 30℃, and dry for 24~72 hours. After drying, demold the microneedles to obtain a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes.

[0059] Example 2 This embodiment provides a method for preparing a multifunctional hybrid microneedle based on a highly efficient NMN@Cu / CeO2 nanozyme delivery system, comprising the following steps: Step 1: Add methacryloyl gelatin (GelMA) to deionized water under a water bath heating condition of 60°C to obtain a precursor solution; wherein, the concentration of methacryloyl gelatin (GelMA) in the precursor solution is 10%.

[0060] Step 2: The NMN@Cu / CeO2 nanozyme, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite, and the precursor solution are mixed to obtain the microneedle tip precursor solution; wherein, the concentration of NMN@Cu / CeO2 nanozyme in the microneedle tip precursor solution is 100 μg / mL, and the concentration of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite is 0.25%.

[0061] Step 3: Add the microneedle tip precursor solution into the PDMS microneedle mold, maintain it under vacuum for 5 minutes to remove air bubbles from the tip, repeat the defoaming step 2-3 times, and use blue light to irradiate for 30 seconds to crosslink it into a gel, thus obtaining the microneedle tip.

[0062] Step 4: Weigh 200mg of polyvinyl alcohol (PVA) into a 5mL centrifuge tube, add 1mL of deionized water, and dissolve at 50~60℃ for 1h. Then add 100mg of lyophilized phenylboronic acid-grafted polylysine (EPL-PBA) to the solution, mix thoroughly until homogeneous, and obtain the microneedle substrate precursor solution after complete dissolution.

[0063] Step 5: Centrifuge the microneedle substrate precursor solution to remove bubbles, then add an appropriate amount of precursor solution to the PDMS mold, place the mold in an oven to dry, maintain the drying temperature at 30℃, and dry for 24~72 hours. After drying, demold the microneedles to obtain a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes.

[0064] Example 3 This embodiment provides a method for preparing a multifunctional hybrid microneedle based on a highly efficient NMN@Cu / CeO2 nanozyme delivery system, comprising the following steps: Step 1: Add methacryloyl gelatin (GelMA) to deionized water under a water bath heating condition of 60°C to obtain a precursor solution; wherein, the concentration of methacryloyl gelatin (GelMA) in the precursor solution is 15%.

[0065] Step 2: The NMN@Cu / CeO2 nanozyme, the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite, and the precursor solution are mixed to obtain the microneedle tip precursor solution; wherein, the concentration of NMN@Cu / CeO2 nanozyme in the microneedle tip precursor solution is 100 μg / mL, and the concentration of photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite is 0.25%.

[0066] Step 3: Add the microneedle tip precursor solution into the PDMS microneedle mold, maintain it under vacuum for 5 minutes to remove air bubbles from the tip, repeat the defoaming step 2-3 times, and use blue light to irradiate for 30 seconds to crosslink it into a gel, thus obtaining the microneedle tip.

[0067] Step 4: Weigh 200mg of polyvinyl alcohol (PVA) into a 5mL centrifuge tube, add 1mL of deionized water, and dissolve at 50~60℃ for 1h. Then add 150mg of lyophilized phenylboronic acid-grafted polylysine (EPL-PBA) to the solution, mix thoroughly until homogeneous, and obtain the microneedle substrate precursor solution after complete dissolution.

[0068] Step 5: Centrifuge the microneedle substrate precursor solution to remove bubbles, then add an appropriate amount of precursor solution to the PDMS mold, place the mold in an oven to dry, maintain the drying temperature at 30℃, and dry for 24~72 hours. After drying, demold the microneedles to obtain a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes.

[0069] Example 4 Unlike Example 1, the concentration of NMN@Cu / CeO2 nanozyme in step 2 was replaced with a concentration of 80 μg / mL to obtain NMN@Cu / CeO2-MN microneedles.

[0070] Example 5 Unlike Example 2, step 3, which involves irradiating the needle with blue light for 30 seconds to crosslink into a gel, was replaced with irradiating the needle with blue light for 45 seconds to crosslink into a gel, resulting in NMN@Cu / CeO2-MN microneedles.

[0071] Example 6 Unlike Example 3, the drying temperature in step 3 was kept at 35°C instead of 30°C, resulting in NMN@Cu / CeO2-MN microneedles.

[0072] 1. Sample preparation This invention provides various microneedle patches and their corresponding preparation methods, including NMN@Cu / CeO2-MN microneedles, MN hydrogel, Cu / CeO2-MN microneedles, and NMN-MN microneedles, and uses them as samples for corresponding tests. Unless otherwise specified, the addition amount and other parameters in the following microneedle preparation methods are the same.

[0073] 1.1 The preparation process of NMN@Cu / CeO2-MN microneedles is as described in the above example.

[0074] The preparation process of the 1.2MN hydrogel is as follows: Step 1: Add methacryloyl gelatin (GelMA) and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate to deionized water under water bath heating conditions to obtain microneedle tip precursor solution.

[0075] Step 2: Add the microneedle tip precursor solution into the PDMS mold, maintain it under vacuum for 5 minutes to remove air bubbles from the tip, and irradiate it with blue light for 30-60 seconds to crosslink it into a gel, thus obtaining the microneedle tip.

[0076] Step 3: Add polyvinyl alcohol (PVA) and phenylboronic acid-grafted polylysine (EPL-PBA) to deionized water under water bath heating conditions to obtain microneedle substrate precursor solution.

[0077] Step 4: Add an appropriate amount of microneedle substrate precursor solution to the PDMS mold, and place the mold in an oven to dry for 24-72 hours. Finally, demold to obtain MN microneedles.

[0078] The preparation process of 1.3Cu / CeO2-MN microneedles is as follows: Step 1: Add methacryloyl gelatin (GelMA), the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and Cu / CeO2 nanozyme (concentration of 100 μg / mL) to deionized water under water bath heating to obtain microneedle tip precursor solution.

[0079] Step 2: Add the microneedle tip precursor solution into the PDMS mold, maintain it under vacuum for 5 minutes to remove air bubbles from the tip, and irradiate it with blue light for 30-60 seconds to crosslink it into a gel, thus obtaining the microneedle tip.

[0080] Step 3: Add polyvinyl alcohol (PVA) and phenylboronic acid-grafted polylysine (EPL-PBA) to deionized water under water bath heating conditions to obtain microneedle substrate precursor solution.

[0081] Step 4: Add an appropriate amount of microneedle substrate precursor solution to the PDMS mold, and place the mold in an oven to dry for 24-72 hours. Finally, demold to obtain Cu / CeO2-MN microneedles.

[0082] The preparation process of 1.4NMN-MN microneedles is as follows: Step 1: Add methacryloyl gelatin (GelMA), photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate, and NMN (concentration 200 μM) to deionized water under water bath heating to obtain microneedle tip precursor solution.

[0083] Step 2: Add the microneedle tip precursor solution into the PDMS mold, maintain it under vacuum for 5 minutes to remove air bubbles from the tip, and irradiate it with blue light for 30-60 seconds to crosslink it into a gel, thus obtaining the microneedle tip.

[0084] Step 3: Add polyvinyl alcohol (PVA) and phenylboronic acid-grafted polylysine (EPL-PBA) to deionized water under water bath heating conditions to obtain microneedle substrate precursor solution.

[0085] Step 4: Add an appropriate amount of microneedle base precursor solution to the PDMS mold, and place the mold in an oven to dry for 24-72 hours. Finally, demold to obtain NMN-MN microneedles.

[0086] 2. Performance Testing and Results The performance of NMN@Cu / CeO2-MN was explored using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (1H NMR), swelling analysis, and NMN release testing.

[0087] 2.1 Microneedle morphology test: like Figure 1 As shown: The microneedle MN patch consists of a 15×15 array, with a needle tip height of 650um, a needle tip distance of 700um, and a microneedle area of ​​approximately 144mm². 2 Scanning electron microscopy (SEM) revealed that the MN needle tip had a complete morphology, with a sharp tip and a rough surface.

[0088] like Figure 2 As shown, the tip of the microneedle is encapsulated with DIL and the substrate is encapsulated with FITC. When the microneedle is photographed under a 3D fluorescence microscope, it can be seen that the microneedle morphology is well maintained.

[0089] 2.2 Microneedle swelling performance test: The results are as follows Figure 3The statistical chart of microneedle swelling performance test shows that, expressed as mean ± SD (n=3), the swelling rates of the MN patches reached 53.40% and 52.06% respectively after 72 hours. The incorporation of composite nanoparticles did not significantly affect the swelling performance of MN. On the one hand, the needle tip absorbs water, softens, and dissolves. Based on the porous hydrophilic structure of the GelMA needle tip, it can effectively absorb interstitial fluid, providing an opportunity for material exchange between MN and the skin, which is conducive to the effective release of composite nanoparticles into the tissue. At the same time, the EPL in the microneedle substrate carries a positive charge, which may form an electrostatic interaction with the negatively charged carboxyl groups in the tissue, further improving the bioadhesion strength of the microneedles after absorbing water.

[0090] 2.3 Fourier Transform Infrared Spectroscopy (FT-IR) Test: The FT-IR spectra of NMN@Cu / CeO2-MN lyophilized microneedles were obtained on an FT-IR spectrometer (Nicolet 6700, Thermo Scientific) from 550 to 4000 cm⁻¹. -1 The study was conducted to investigate the chemical composition.

[0091] Test results are as follows Figure 4 As shown, FTIR analysis was performed to confirm the successful loading of NMN@Cu / CeO2 into the microneedle substrate PVA / EPL-PBA and the tip. EPL-PBA was analyzed at 1423 cm⁻¹. -1 An absorption band for the BO bond in group B(OH)₂ is shown at [value missing]. Two characteristic bands were identified in PVA / EPL-PBA: BO bond (1390 cm⁻¹). -1 ) and BOC bond (1160 cm) -1 This indicates that dynamic borate bonds have formed within the microneedle matrix. Furthermore, FTIR spectroscopy reveals a 2350 cm⁻¹... -1 and 2852 cm -1 The characteristic peaks at the location confirm the successful incorporation of NMN@Cu / CeO2.

[0092] 2.4 Liquid NMR 1H spectrophotometry analysis: The results are as follows Figure 5As shown: The materials were characterized by 1H NMR spectroscopy, verifying the successful synthesis of GelMA and EPL-PBA. First, the carbon-carbon double bond signals of methacrylate appeared at 5.2 ppm and 5.6 ppm, confirming that methacrylic anhydride had been successfully grafted onto the gelatin chain (Figure B). Similarly, the peaks of EPL-PBA at 1.68, 1.42, 1.32, and 3.07 ppm correspond to protons on the EPL-PBA backbone. The peak at 4.41 ppm is attributed to a proton on the carbon adjacent to the newly formed amide bond, while the peak between 7.94 and 7.43 ppm is attributed to an aromatic proton on the benzene ring (Figure A).

[0093] 2.5 Microneedle in vitro drug release assay: Rhodamine B, a model drug, was used to demonstrate drug release behavior in microneedles. Fluorescently labeled nanoparticles were obtained by incubating RB with composite nanoparticles, and then loaded into microneedles to prepare microneedle patches containing RB (1 mg / mL). The microneedles were immersed in 5 mL of PBS (pH 7.4) at 37°C, and the absorbance of fluorescence in the supernatant was measured at specified time points (0, 1, 3, 6, 12, 24, 48, 72, 96, and 120 h). The cumulative release in the PBS solution was obtained based on a standard fluorescence intensity-concentration curve.

[0094] The results are as follows Figure 6 As shown, in order to evaluate the drug loading and release behavior of NMN@Cu / CeO2, we used RhB as a model drug to load into MN. The MN patch loaded with RhB was immersed in phosphate buffered saline (PBS). The quantitative analysis results based on the standard curve of RhB solution showed that the drug was released slowly and continuously in MN, and the release efficiency reached 73.14% on day 6.

[0095] 2.6 In vitro cell compatibility test: Human fibroblasts (HFB) and human umbilical vein endothelial cells (HUVECs) were incubated in DMEM medium (Gibco) supplemented with 10% FBS and 1% penicillin and streptomycin (Hyclone) at 37°C in an incubator with 5% CO2. (Using LIVE / DEAD) ®The cytotoxicity of microneedles was studied using an activity / cytotoxicity kit (YESAN, Shanghai, China). HFB cells (or HUVEC cells) were seeded at a density of 10,000 cells / well in 96-well plates, and microneedle patches were co-cultured with HFB (or HUVEC) for 24 h. The LIVE / DEAD® kit was used to assess the viability of cells co-cultured with microneedle patches. After 24 hours of co-culture, the microneedle patches and culture medium were removed, and LIVE / DEAD® reagent was added according to the instructions. After a further 45 minutes of incubation, images were taken using a fluorescence microscope (Nikon, DS-Ri2, Japan).

[0096] When HFB (or HUVEC) is co-cultured with microneedle patches, and the live / dead assay is used to visualize cell morphology, the results are as follows: Figure 7 and Figure 8 As shown, most HFB (or HUVEC) cells were stained green (live cells), and only a small number of cells were stained red (dead cells), demonstrating the high cell compatibility of NMN@Cu / CeO2-MN microneedles.

[0097] 2.7 In vitro ROS scavenging ability test: The 2',7'-dichlorofluorescein diacetate (DCFH-DA, Maokang, Shanghai, China) probe was used to evaluate the intracellular ROS scavenging ability of microneedles. In short, HUVECs were seeded in 6-well plates and ROS was induced by ROS up (MKbio, Shanghai, China), followed by co-culturing with microneedle samples for 12 hours. A negative control (PBS) was provided. Afterward, the microneedles were removed, and DCFH-DA was added for staining at 37°C for 20 minutes. ROS scavenging ability was measured in each group using flow cytometry (BD Accuri® C6, USA), with each group repeated three times.

[0098] The results are as follows Figure 9 As shown, ROS-positive cells labeled with DCFH-DA and ROS inducers significantly promoted ROS production, while microneedles effectively mitigated ROS production, with the most significant reductions observed in the NMN@MN and NMN@Cu / CeO2-MN groups. This is likely due to the introduction of NMN, a potent antioxidant and sirtuin agonist. During microneedle preparation, NMN@Cu / CeO2 was introduced into the needle tip, and its scavenging of ∙OH free radicals generated by hydrogen peroxide endows the microneedles with excellent ROS scavenging capabilities, contributing to wound healing. In summary, these findings highlight the potential of NMN@Cu / CeO2-MN to reduce excessive oxidative stress and protect cells in the microenvironment of diabetic wounds.

[0099] 2.8 In vitro antibacterial performance test: The antibacterial properties of the microneedles were determined using the plate count method. In short, 10 µL of a suspension of *E. coli* and *S. aureus* (10 µL of plate count) was used to determine the antibacterial properties. 8 CFU / mL was added to the surface of MN, Cu / CeO2-MN, NMN-MN, and NMN@Cu / CeO2-MN, and incubated in 48-well plates at 37°C for 2 hours. Then, 1 mL of PBS was introduced into each well to resuspend any bacterial survivors. Finally, 10 µL of the resuspended solution was added to an agar plate, and colonies were counted after incubation at 37°C for 24 hours. Antibacterial rate (%) = (Bacterial count in control - Survival count on microneedles) / Number of bacteria in control × 100%.

[0100] Figure 10 Images show the contact antimicrobial activity of microbes treated with PBS, MN, Cu / CeO2-MN, NMN-MN, and NMN@Cu / CeO2-MN against Escherichia coli and Staphylococcus aureus, respectively. Antimicrobial dressings can minimize infection by harmful microorganisms and improve delayed wound healing, a key challenge in the healing process of diabetic wounds.

[0101] Compared to PBS, Cu / CeO2-MN and NMN@Cu / CeO2-MN exhibited bactericidal activity exceeding 99% against *E. coli*, while MN and NMN-MN achieved bactericidal rates of 85% and 89%, respectively. The antibacterial activity of MNs is primarily attributed to the successful incorporation of EPL, a macromolecule renowned for its antibacterial properties. The positive charge of EPL interacts with the negatively charged phospholipid membrane of bacteria, leading to membrane rupture and bacterial death, thus providing excellent infection control. Similarly, Cu / CeO2-MN and NMN@Cu / CeO2-MN achieved bactericidal rates of approximately 72.3% and 74.2% against MRSA, respectively, higher than the bactericidal rates of the MN and NMN-MN groups, which were 53.2% and 49.7%, respectively. The incorporation of the composite nanomaterials enhanced the antibacterial activity of MN, likely due to the presence of Cu-doped CeO2, which disrupts the bacterial membrane and alters protein or enzyme structures. In conclusion, NMN@Cu / CeO2-MN demonstrates significantly enhanced antibacterial capabilities, showing great potential for addressing diabetic wound infections. There are three potential mechanisms by which metal ions limit microbial growth: First, metal ions can directly disrupt ATP synthesis and DNA replication by crossing the cell membrane; second, Mg... 2+Electrostatic forces may fix cell membranes, impairing cell integrity and preventing protons and other molecules from effectively passing through them. Finally, metal ions may induce oxidative stress by generating reactive oxygen species (ROS), which can disrupt normal bacterial activity and ultimately kill bacteria by damaging their membranes, DNA, and mitochondria. The combination of all these properties significantly enhances the antibacterial activity of the hydrogel, suggesting the great potential of microneedles in protecting diabetic wounds from contamination.

[0102] 2.9 In vivo hemostasis test: A mouse model of hemorrhagic liver and tail hemorrhage was established. Mice were anesthetized and then fixed on a surgical table. The liver of the rats was exposed through an abdominal incision, and filter paper was weighed and placed under the liver. Then, a 5 mm deep bleeding wound was introduced into the liver with a blade, and microneedles were immediately applied to the bleeding site. The mouse tail was severed with scissors 3 cm from the tip, and microneedles were then applied to the severed site. The control group was designated as the untreated group. The filter paper was then weighed, and the amount of blood loss was calculated.

[0103] Wound healing typically involves stages such as hemostasis, inflammation, tissue formation, and remodeling. Hemostasis is the first step in wound healing, therefore hemostatic dressings are crucial for controlling the bleeding phase. To evaluate the hemostatic performance of NMN@Cu / CeO2-MN, mouse tail hemorrhage and liver hemorrhage models were used. MN was applied to the severed tail, and blood loss was recorded. Figure 11 As shown, NMN@Cu / CeO2-MN significantly reduced blood loss compared to the control group. Quantitative analysis revealed that the tail blood loss in the NMN@Cu / CeO2-MN group was 12.87 mg, while that in the control group was 52.69 mg.

[0104] Furthermore, a mouse model of acute liver hemorrhage was established. When NMN@Cu / CeO2-MN was applied to a liver wound, it significantly reduced bleeding. Hemostatic quantification showed that blood loss in the MN group was 19.37 mg, significantly lower than the 59.03 mg in the control group. The hemostatic mechanism of NMN@Cu / CeO2-MN may involve multiple factors. First, the needle tip (GelMA) and base (EPL-PBA) absorb interstitial fluid, adhere to the tissue surface, and provide a physical barrier to reduce bleeding. Second, GelMA promotes platelet adhesion and aggregation, contributing to the coagulation process. Simultaneously, the microneedle patch releases and exposes NMN@Cu / CeO2 to the wound site, due to platelets reacting with positively charged amino groups and Cu... 2+Specific binding and bioactivation of the coagulation process accelerate hemostasis. In summary, the hemostatic mechanism of NMN@Cu / CeO2-MN promotes hemostasis through a combination of physical occlusion and bioactivation. The needle tip inserted into the tissue expands and softens due to rapid fluid absorption, forming a three-dimensional network structure that acts as a physical blockage of the wound. The released composite nano-activated biocoagulation process accelerates hemostasis. NMN@Cu / CeO2-MN possesses excellent antibacterial and hemostatic properties, making it a promising candidate for clinical applications in diabetic wound management.

[0105] 2.10 Diabetic Wound Healing Test: First, C57 mice were fed a high-fat, high-sugar diet for four weeks, followed by five days of streptozotocin (STZ, 10 mg / kg; Meilunbio, China). A blood glucose level above 16.7 mM was considered type 2 diabetes. Two weeks later, the mice were randomly assigned to five groups (n=12) as a control (Tegaderm Film, 3M, USA), MN, Cu / CeO2-MN, NMN-MN, and NMN@Cu / CeO2-MN. A full-thickness wound was then created in the center of the back using an 8 mm biopsy punch. Next, 150 μL of hydrogel or PBS (control) was injected into the wound site, which was then covered with a Tegaderm membrane (3M, USA). The wound area was measured and photographed using a digital camera on day 0, day 3 (inflammatory phase), day 7 (proliferative phase), and day 14 (remodeling phase).

[0106] In mice, an 8 mm diameter wound was created on the dorsal side using a skin punch, followed by treatment with MN, Cu / CeO2-MN, NMN-MN, and NMN@Cu / CeO2-MN, with Tegaderm dressing serving as a control. Wound size was recorded on days 0, 3, 7, and 14. Visual inspection showed significant reduction in wound size in all groups, with the NMN@Cu / CeO2-MN group healing the fastest and the control group the slowest. Quantitative analysis of the wound area further assessed wound closure, indicating that on day 3, the NMN@Cu / CeO2-MN group exhibited the greatest wound contraction compared to the other treatment groups. Figure 13 (P<0.05). On day 7, the wound area in the NMN@Cu / CeO2-MN group was significantly reduced (21.25%), while the wound areas in the control group, MN, Cu / CeO2-MN, and NMN-MN groups were 54.17%, 41.81%, 34.33%, and 27.17%, respectively. All microneedle-treated wounds were more robust than those treated with the Tegaderm membrane (control group, P<0.05). By day 14, the wound closure rate in the NMN@Cu / CeO2-MN group reached 95.6%, and the healing rates in the Cu / CeO2-MN and NMN-MN groups were higher than those in the control and MN groups.

[0107] Given that microneedling can enhance wound healing, the number of newly formed blood vessels is a key factor in the effectiveness of the healing process. Immunofluorescence assays were used to measure the expression of CD31, a representative of vascular structure, to estimate the number of blood vessels in newly formed skin tissue on day 7. The highest CD31 expression was observed in the NMN@Cu / CeO2-MN group, indicating a significantly higher number of blood vessels in the microneedling-treated wound compared to the control group. Figure 14 (P<0.05) indicates that microneedles have a stronger pro-angiogenic potential, demonstrating that the NMN@Cu / CeO2-MN group had the greatest angiogenic effect. All results show that the NMN@Cu / CeO2-MN group directly promotes the formation of more blood vessels and, through the combined effect of the hydrogel, prevents excessive oxidative stress and inflammation in vascular endothelial cells, while releasing NMN and Cu / CeO2. Therefore, the NMN@Cu / CeO2-MN group's ability to scavenge ROS, reduce inflammation, and promote angiogenesis can accelerate the healing of diabetic wounds.

[0108] Unlike most current dressings that target only one stage of diabetic wound healing, the NMN@Cu / CeO2-MN group developed in this invention has been shown to promote healing during the hemostatic, inflammatory, and proliferative phases of wound repair. Thanks to NMN, the microneedles can significantly reduce ROS to achieve anti-inflammatory effects. Furthermore, the inherent antibacterial properties of QCS and Cu / CeO2 synergistically enhance the antibacterial efficacy of the hydrogel. This composite hydrogel can significantly promote fibroblast survival and greatly accelerate the healing of diabetic wounds by clearing ROS, enhancing angiogenesis, and reducing inflammation. These results indicate that NMN@Cu / CeO2-MN microneedles are easy to prepare and apply, showing great promise for future clinical applications as a diabetic wound dressing.

[0109] In summary, this invention provides a multifunctional hybrid microneedle based on efficient delivery of NMN@Cu / CeO2 nanozymes. This microneedle has good tissue adhesion, excellent biocompatibility, hemostasis, ROS scavenging, angiogenesis promotion and antibacterial ability, and can effectively accelerate the healing of diabetic wounds by continuously delivering drugs to the wound area.

[0110] The above content is only for illustrating the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. Any modifications made to the technical solution based on the technical concept proposed in this invention shall fall within the scope of protection of the claims of this invention.

Claims

1. A method for preparing a multifunctional hybrid microneedle based on a highly efficient NMN@Cu / CeO2 nanozyme delivery system, characterized in that, Includes the following steps: Under water bath heating conditions, freeze-dried methacryloyl gelatin was added to deionized water to prepare a precursor solution. NMN@Cu / CeO2 nanozyme and photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonate were mixed with the precursor solution to obtain microneedle tip precursor solution. Under water bath heating conditions, polyvinyl alcohol and polylysine grafted with phenylboronic acid were added to deionized water to obtain a microneedle substrate precursor solution. The microneedle tip precursor solution was added to the microneedle mold for vacuum defoaming and concentration, then blue light curing and crosslinking were performed. The microneedle base precursor solution was then added to the microneedle mold and dried and demolded to obtain a multifunctional hybrid microneedle based on the efficient delivery of NMN@Cu / CeO2 nanozymes.

2. The method for preparing multifunctional hybrid microneedles based on efficient delivery of NMN@Cu / CeO2 nanozymes according to claim 1, characterized in that, The mass ratio of lyophilized methacryloyl gelatin, NMN@Cu / CeO2 nanozyme, and deionized water in the precursor solution is (50~150):(0.08~0.12):1; the concentration of NMN@Cu / CeO2 nanozyme in the microneedle tip precursor solution is 80~120 μg / mL; and the concentration of the photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphonite in the microneedle tip precursor solution is 0.25%.

3. The method for preparing multifunctional hybrid microneedles based on efficient delivery of NMN@Cu / CeO2 nanozymes according to claim 1, characterized in that, The concentration of polyvinyl alcohol in the microneedle substrate precursor solution is 200 mg / mL, and the concentration of phenylboronic acid-grafted polylysine is 50-150 mg / mL.

4. The method for preparing multifunctional hybrid microneedles based on efficient delivery of NMN@Cu / CeO2 nanozymes according to claim 1, characterized in that, The preparation method of the NMN@Cu / CeO2 nanozyme is as follows: Cu / CeO2 nanoparticles were suspended in a first NaOH solution and stirred. Then, C3H5Cl and a second NaOH solution were added sequentially and stirred overnight at room temperature to obtain the first reaction product. The first reaction product was centrifuged and washed until the first supernatant of the first reaction product was neutral. Then, the first supernatant was vacuum dried to obtain the dried product. The dried product and polyethyleneimine (PEI) were dissolved in deionized water and stirred to obtain the second reaction product. The second reaction product was centrifuged and washed until the second supernatant of the second reaction product was neutral. Then, it was dialyzed and centrifuged in sequence to remove the supernatant and then freeze-dried to obtain PEI-modified Cu / CeO2. The PEI-modified Cu / CeO2 and NMN aqueous solution were then mixed and incubated at room temperature with stirring. After centrifugation and washing to remove the supernatant, NMN@Cu / CeO2 nanozyme was prepared and resuspended in deionized water for storage.

5. The method for preparing multifunctional hybrid microneedles based on efficient delivery of NMN@Cu / CeO2 nanozymes according to claim 4, characterized in that, The concentration of the first NaOH solution is 0.1M; the concentration of the second NaOH solution is 2M; the ratio of the Cu / CeO2 nanoparticles, the first NaOH solution, C3H5Cl, and the second NaOH solution is 50mg:10mL:10mL:0.5mL; The concentration of polyethyleneimine (PEI) is 0.004 M; the ratio of the dried product, PEI, and deionized water is 50 mg: 40 mg: 250 mL. The concentration of the NMN aqueous solution is 0.0075M; the ratio of the amount of PEI-modified Cu / CeO2 and NMN aqueous solution is 50mg:1mL.

6. The method for preparing multifunctional hybrid microneedles based on efficient delivery of NMN@Cu / CeO2 nanozymes according to claim 1, characterized in that, The method for preparing the phenylboronic acid-grafted polylysine is as follows: 2-(N-morpholino)ethanesulfonic acid (MES) was dissolved in deionized water and mixed thoroughly until the solution was clear and transparent. Then, polylysine (EPL), phenylboronic acid (PBA), N,N-dimethylformamide (DMF), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) were added to the solution in sequence, and the mixture was stirred overnight at 60°C. After the solution returned to room temperature, the supernatant was collected by centrifugation. The supernatant was dialyzed with deionized water, and finally, the dialyzed solution was freeze-dried to obtain phenylboronic acid-grafted polylysine.

7. The method for preparing multifunctional hybrid microneedles based on efficient delivery of NMN@Cu / CeO2 nanozymes according to claim 6, characterized in that, The ratio of 2-(N-morpholino)ethanesulfonic acid (MES), deionized water, polylysine (EPL), phenylboronic acid (PBA), N,N-dimethylformamide (DMF), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and N-hydroxysuccinimide (NHS) is 2g:200mL:10g:2.64g:20mL:6.1g:3.66g.

8. The method for preparing multifunctional hybrid microneedles based on efficient delivery of NMN@Cu / CeO2 nanozymes according to claim 1, characterized in that, The water bath heating temperature is 50~60℃; the vacuum defoaming and concentration treatment is performed 2~3 times; the blue light curing and crosslinking time is 30~60s; the drying time is 24~72h, and the drying temperature is 30~35℃.

9. A multifunctional hybrid microneedle based on efficient delivery of NMN@Cu / CeO2 nanozymes, characterized in that, The multifunctional hybrid microneedles based on the efficient delivery of NMN@Cu / CeO2 nanozymes, as described in any one of claims 1 to 8, were prepared.

10. The multifunctional hybrid microneedles based on the efficient delivery of NMN@Cu / CeO2 nanozymes, prepared by the method according to any one of claims 1 to 8, are used in the preparation of wound dressings for the treatment of diabetes.

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