A bactericidal and anti-inflammatory multi-layer microneedle patch with programmed response and a preparation method thereof

Abstract

The application discloses a bactericidal and anti-inflammatory multi-layer microneedle patch with programmed response and a preparation method, and relates to the technical field of biomedical materials and wound repair. The microneedle patch comprises an outer shell layer, a core layer and a sandwich layer, the sandwich layer wraps the core layer, and the outer shell layer is coated outside the needle body of the sandwich layer. The component of the core layer is PVP and PVA loaded curcumin nanoparticles. The component of the outer shell layer is PVP and PVA loaded copper chloride. The sandwich layer is an enteric coating. The preparation method adopts reverse combination from solid to liquid, the solidified inner core is used as a male die, and is directly added to an un-solidified solution in a female die to form a mechanical interlocking structure. The application realizes layered time-sequencing drug delivery under multi-signal response, has good antibacterial, anti-inflammatory and wound healing abilities, and good mechanical properties, biocompatibility and large-scale preparation potential, and can significantly improve the antibacterial and repair efficiency of chronic wounds.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials and wound repair technology, specifically to a programmed response antibacterial and anti-inflammatory multilayer microneedle patch and its preparation method. Background Technology

[0002] Chronic wounds are often slow to heal and are frequently accompanied by persistent biofilm-related infections and abnormal inflammatory responses. The healing process is influenced by multiple factors, including the biofilm barrier, bacterial residue, local acidic environment, oxidative stress, and insufficient tissue repair capacity. Biofilms, composed of extracellular polymeric matrix secreted by bacteria, significantly reduce the penetration of antimicrobial drugs and the efficiency of host immune clearance, leaving bacteria in a persistent state that is difficult to eradicate and further exacerbating the local inflammatory response. Furthermore, the acidic microenvironment formed within the biofilm not only weakens the activity of antimicrobial agents but also inhibits the migration and proliferation of fibroblasts and keratinocytes, thereby delaying tissue repair.

[0003] Clinical treatment often relies on a combination of phased strategies, including debridement, antibacterial treatment, and promoting healing. However, these methods still have limitations in clearing biofilms and regulating local inflammation. For example, frequent debridement may damage newly formed tissue; long-term use of antibiotics can lead to the proliferation of drug-resistant bacteria and disrupt the local microecology; and persistent high-level inflammatory responses inhibit tissue regeneration. Therefore, how to effectively inhibit bacteria while avoiding excessive damage to host cells, and achieve dynamic regulation of inflammation and repair processes, has become a key issue in the treatment of chronic wounds.

[0004] In recent years, antibacterial methods based on reactive oxygen species (ROS) have attracted attention due to their ability to disrupt bacterial structures and interfere with biofilms. However, ROS are also highly oxidizing, and their excessive accumulation can lead to oxidative damage and increased inflammation in host cells, thereby delaying the healing process. To achieve antioxidant and anti-inflammatory effects without excessive damage to host cells, ROS must be eliminated. This leads to mutual interference between antibacterial and antioxidant / anti-inflammatory approaches. Therefore, precise spatial and temporal control of ROS levels during the antibacterial process is particularly important.

[0005] Microneedle drug delivery systems can penetrate the skin barrier through micron-sized needles to deliver drugs directly to the epidermis or dermis, thereby improving local drug utilization and enhancing penetration through biofilms. However, traditional microneedles are mostly passive release systems, making it difficult to adjust the delivery according to changes in the wound microenvironment, such as pH, ROS, and inflammation levels at different stages, thus limiting their application in complex chronic wounds. Therefore, developing an intelligent microneedle system that can adaptively respond to changes in the wound microenvironment and precisely regulate antibacterial and anti-inflammatory processes is of great significance for promoting efficient healing of chronic wounds. Summary of the Invention

[0006] This invention addresses the technical problems of traditional microneedle drug delivery systems, such as their inability to adjust according to changes in the microenvironment (pH, ROS, inflammation levels, etc.) at different stages of a wound, and the mutual interference between antibacterial, antioxidant, and anti-inflammatory effects. The aim is to provide a multi-layered microneedle patch with programmed response for sterilization and anti-inflammation, along with its preparation method. This patch achieves layered, sequential drug delivery under multi-signal responses, exhibiting excellent antibacterial, anti-inflammatory, and wound-healing capabilities, as well as good mechanical properties, biocompatibility, and potential for large-scale production. It can significantly improve the antibacterial and repair efficiency of chronic wounds.

[0007] The present invention is achieved through the following technical solution.

[0008] The first objective of this invention is to provide a multilayer microneedle patch with programmed response for sterilization and anti-inflammation. The microneedle patch has a layered structure with a plurality of needles, including an outer shell layer, a core layer and a sandwich layer. The sandwich layer encloses the core layer, and the outer shell layer covers the needles of the sandwich layer.

[0009] The core layer consists of polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and curcumin nanoparticles coated with ZIF-8 (zeolite imidazole acid framework-8), with a mass ratio of 1:(0.9-1.1):(1.1-1.5).

[0010] The outer shell consists of polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and copper chloride, in a mass ratio of 1:(0.9-1.1):(0.11-0.15).

[0011] The sandwich layer is an enteric coating. In one specific embodiment of the present invention, the enteric coating is Eudragit S100.

[0012] The technical principle is as follows:

[0013] The multilayer microneedle patch of this invention adopts a three-layer structure of core-sandwich-shell. The introduction of the enteric coating sandwich layer eliminates functional crosstalk caused by solvent erosion and component diffusion, achieving sequential response based on the wound microenvironment, which is difficult to achieve with traditional microneedles: in an acidic environment, the outer shell layer rapidly dissolves and releases Cu. 2+ It generates active free radicals to achieve early antibacterial effects; as the pH rises to neutral, the sandwich layer dissolves to expose the core layer, and the structural stability of ZIF-8 allows curcumin to be released in a delayed manner during this stage, avoiding interference with the bactericidal effect of reactive oxygen species; under the condition of increased ATP leakage during bacterial death, the core layer (Cur@ZIF-8) is triggered to decompose and release curcumin, which is used for later anti-oxidation, anti-inflammatory and tissue repair, and can reduce tissue damage.

[0014] Therefore, the multilayer microneedle structure of this invention can penetrate biological membranes and directly deliver drugs to the site of infection, thereby enhancing drug delivery penetration and local effectiveness. Through multi-signal responses such as pH and ATP, it achieves sequential release of antibacterial, antioxidant, and repair-promoting effects, realizing programmed treatment triggered by the microenvironment. This phased regulation of inflammation and regeneration processes helps accelerate the transition of the wound towards the proliferative and remodeling phases. The delayed release of curcumin avoids its initial removal of ROS, which would affect the bactericidal effect, and overcomes the problem of mutual interference between antibacterial and antioxidant effects.

[0015] In summary, the multilayer microneedle structure of this invention can adaptively respond to changes in the wound microenvironment, achieve precise control of antibacterial and anti-inflammatory processes, reduce the risk of bacterial resistance and host tissue damage, and improve the overall repair efficiency of chronic wounds.

[0016] Furthermore, the ZIF-8-coated curcumin nanoparticles have an average particle size of 120-140 nm, a drug loading of more than 10%, and an encapsulation efficiency of more than 50%.

[0017] Furthermore, the needles on the surface of the microneedle patch are arranged in an array.

[0018] Furthermore, the length of a single needle is 800-900μm, the diameter of the bottom of the needle is 400-450μm, and the distance between the tips of two adjacent needles is 600-800μm.

[0019] Preferably, the core layer comprises polyvinylpyrrolidone, polyvinyl alcohol, and ZIF-8-coated curcumin nanoparticles in a mass ratio of 1:1:1.3; the outer shell comprises polyvinylpyrrolidone, polyvinyl alcohol, and copper chloride in a mass ratio of 1:1:0.13.

[0020] The second objective of this invention is to provide a method for preparing a programmed responsive, antibacterial, and anti-inflammatory multilayer microneedle patch, comprising the following steps:

[0021] Add the core layer microneedle precursor solution to the microneedle mold, centrifuge and dry to obtain the microneedle core layer Cur@ZIF-8-MN;

[0022] Add the sandwich layer microneedle precursor liquid to the microneedle mold, centrifuge and dry. Use the microneedle core layer Cur@ZIF-8-MN obtained in the previous step as a positive mold, press it into the mold filled with the sandwich layer microneedle precursor liquid, dry it, add the sandwich layer microneedle precursor liquid for encapsulation, and dry it to obtain the enteric-coated double-layer microneedles CZ@EC-MN.

[0023] Add the outer shell layer microneedle precursor liquid to the microneedle mold, centrifuge and dry. Use the bilayer microneedle CZ@EC-MN obtained in the previous step as a positive mold, press it into the mold filled with the outer shell layer microneedle precursor liquid, and dry to obtain the multilayer microneedle patch CZ@EC@Cu-MN.

[0024] In the existing technology, the traditional synthesis methods for multilayer core-shell microneedles mainly include layer-by-layer casting centrifugation and molding. Layer-by-layer casting centrifugation is a simple stacking of components, which often results in the synthesized microneedles not having a regular core-shell structure but a layered stacked structure due to component limitations. Molding requires multiple demolding operations, which often results in problems such as hollow shells being difficult to demold and shell cracking.

[0025] The multilayer microneedle synthesis method described in this invention employs a reverse solid-to-liquid approach. The solidified core (core layer / sandwich layer) serves as the male mold, directly added to the unsolidified solution in the female mold. The tip of the male mold, like a "key," inserts into the unsolidified liquid in the female mold, forming a mechanical interlocking structure. The unsolidified components of the female mold fully encapsulate the solidified male mold components, allowing the female mold components to be fully encapsulated and molded in situ. This method ensures the integrity of the needle shape between layers, preventing easy peeling and interlayer slippage during use. It guarantees the overall mechanical integrity of the microneedle patch, endowing the microneedles with stronger mechanical properties, achieving a mechanical strength of approximately 4.1 N / needle. Simultaneously, it significantly simplifies the preparation process, reduces the difficulty of preparing multilayer microneedles, and improves the yield.

[0026] Furthermore, the preparation method of the core layer microneedle precursor fluid is as follows:

[0027] Polyvinylpyrrolidone was added to deionized water in a certain proportion and dispersed evenly. Polyvinyl alcohol was then slowly added and stirred. ZIF-8-coated curcumin nanoparticles were added and ultrasonically dispersed to obtain the core layer microneedle precursor fluid.

[0028] Furthermore, the method for preparing the ZIF-8-coated curcumin nanoparticles is as follows:

[0029] Zinc nitrate hexahydrate was dissolved in water to prepare an aqueous solution of zinc nitrate. 2-methylimidazole and curcumin were then dispersed sequentially into a methanol solution to form a homogeneous mixed precursor liquid.

[0030] The two liquids were rapidly mixed, subjected to ultrasonic and stirring treatment, and after the reaction was completed, they were washed and dried to obtain Cur@ZIF-8 nanoparticles.

[0031] The mass ratio of zinc nitrate hexahydrate, 2-methylimidazole, and curcumin is 30:(60-70):(1-3).

[0032] Furthermore, the preparation method of the sandwich layer microneedle precursor fluid is as follows:

[0033] Enteric coating was added to ethanol, and the mixture was stirred and ultrasonically dispersed to obtain a sandwich microneedle precursor solution.

[0034] Furthermore, the preparation method of the outer shell microneedle precursor fluid is as follows:

[0035] Add vinylpyrrolidone to deionized water in a certain proportion, mix well, add copper chloride to dissolve completely, then slowly add polyvinyl alcohol and stir evenly to obtain the shell layer microneedle precursor solution.

[0036] A third objective of this invention is to provide a wound healing material comprising the aforementioned programmed responsive antibacterial and anti-inflammatory multilayer microneedle patch.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] 1. The multilayer microneedle structure of this invention can penetrate biological membranes and directly deliver drugs to the site of infection, thereby enhancing drug delivery penetration and local effectiveness. Through multi-signal responses such as pH and ATP, it achieves sequential release of antibacterial, antioxidant, and repair-promoting effects, realizing programmed treatment triggered by the microenvironment. This phased regulation of inflammation and regeneration processes helps accelerate the transition of the wound to the proliferative and remodeling phases. The delayed release of curcumin avoids its initial removal of ROS, which would affect the bactericidal effect, and overcomes the problem of mutual interference between antibacterial and antioxidant effects. Therefore, the multilayer microneedle structure of this invention can adaptively respond to changes in the wound microenvironment, achieve precise regulation of antibacterial and anti-inflammatory processes, reduce the risk of bacterial resistance and host tissue damage, and improve the overall repair efficiency of chronic wounds.

[0039] 2. The multilayer microneedle synthesis method of this invention adopts a reverse solid-to-liquid combination approach. The solidified core (core layer / sandwich layer) is used as a male mold and directly added to the unsolidified solution in the female mold. The tip of the male mold, like a "key," is inserted into the unsolidified liquid in the female mold, forming a mechanical interlocking structure. The unsolidified components of the female mold fully encapsulate the solidified male mold components, allowing the female mold components to be fully encapsulated and molded in situ. This method ensures the integrity of the needle shape between layers, preventing easy peeling and interlayer slippage during use. It guarantees the overall mechanical integrity of the microneedle patch, endowing the microneedles with stronger mechanical properties, achieving a mechanical strength of approximately 4.1 N / needle. Simultaneously, it greatly simplifies the preparation process, reduces the difficulty of preparing multilayer microneedles, and improves the yield. Attached Figure Description

[0040] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings:

[0041] Figure 1 This is a schematic diagram of the structure of the multilayer microneedle patch of the present invention;

[0042] Figure 2 This is a schematic diagram of the preparation method of the multilayer microneedle patch of the present invention;

[0043] Figure 3 A scanning electron microscope (SEM) schematic diagram of Cur@ZIF-8 prepared in this invention;

[0044] Figure 4 This is a scanning electron microscope (SEM) schematic diagram of the multilayer microneedle patch prepared according to the present invention.

[0045] Figure 5 The fluorescence detection results of the multilayer microneedle patch prepared in this invention are shown in the image. From right to left, the images are the cross-sectional fluorescence images of the shell layer, the sandwich layer, and the combined image.

[0046] Figure 6 The statistical results of the particle size of Cur@ZIF-8 prepared in this invention are shown in the figure.

[0047] Figure 7 Mechanical property test diagram of the CZ@E@C-MN multilayer microneedle patch prepared in this invention;

[0048] Figure 8 The image shows the activity test results of the CZ@E@C-MN multilayer microneedle patch-like POD prepared in this invention. Figure 8 A represents the POD-like activity of the soaking solution for CZ@E@C-MN multilayer microneedle patches. Figure 8 B is a test diagram of the activity source of multilayer microneedle patch-type POD;

[0049] Figure 9 The figure shows the results of the dual-response release performance of the CZ@E@C-MN multilayer microneedle patch prepared in this invention.

[0050] Figure 10 The image shows the antibacterial performance of the CZ@E@C-MN multilayer microneedle patch prepared in this invention.

[0051] Figure 11 The image shows the biofilm removal results of the CZ@E@C-MN multilayer microneedle patch prepared in this invention;

[0052] Figure 12 The image shows the cell compatibility results of the CZ@E@C-MN multilayer microneedle patch prepared according to the present invention. Figure 12 Figure A shows the statistical results of cell viability after co-incubation of CZ@E@C-MN multilayer microneedle patch soaking solution with cells for 12 h and 24 h. Figure 12 B shows the staining results of live / dead cells after co-incubation of the CZ@E@C-MN multilayer microneedle patch soaking solution with cells for 24 hours;

[0053] Figure 13 The image shows the anti-inflammatory properties of the CZ@E@C-MN multilayer microneedles prepared in this invention.

[0054] Figure 14 The results of the cell migration-promoting performance of the CZ@E@C-MN multilayer microneedles prepared in this invention are shown in the figure. The left figure shows the comparison of cell scratches after 24 h of culture, and the right figure shows the comparison of cell migration ability.

[0055] Figure 15 The figure shows the wound healing performance of the CZ@E@C-MN multilayer microneedles prepared in this invention. Figure 15 Figure A shows the physical changes in wound healing of diabetic mice using CZ@E@C-MN multilayer microneedles. Figure 15 B represents the wound healing rate in diabetic mice.

[0056] Figure 16 The image shows the results of the CZ@E@C-MN multilayer microneedles prepared in this invention promoting the histological changes of wounds in diabetic mice.

[0057] Figure 17 The graph shows the changes in collagen content in wound tissue promoted by the multilayer microneedles of CZ@E@C-MN prepared in this invention. Detailed Implementation

[0058] To make the objectives, technical solutions, and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0059] Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. However, unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions may be omitted. This is to avoid making the following description unnecessarily lengthy and to facilitate understanding by those skilled in the art.

[0060] The "scope" disclosed in this invention is defined in the form of a lower limit and an upper limit. A given scope is defined by selecting a lower limit and an upper limit, which define the boundaries of the specific scope. The scope defined in this way can include or exclude end values, and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a scope.

[0061] Unless otherwise specified, all embodiments and optional embodiments of the present invention can be combined with each other to form new technical solutions.

[0062] Unless otherwise specified, all technical features and optional technical features of this invention can be combined to form new technical solutions.

[0063] Unless otherwise specified, the terms "comprising" and "including" as used in this invention can be open-ended or closed-ended. For example, "comprising" and "including" can mean that other substances not listed may also be included, or that only the listed substances may be included.

[0064] Unless otherwise specified, all steps of the present invention may be performed sequentially or randomly, preferably sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0065] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0066] It should be noted that, unless otherwise specified, the experimental methods used in the embodiments are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0067] Curcumin is a natural polyphenol with antioxidant and anti-inflammatory properties, widely used in cancer treatment and wound healing. During wound healing, a large number of free radicals are generated. Curcumin molecules themselves are powerful free radical scavengers, neutralizing harmful free radicals. Simultaneously, curcumin effectively inhibits key inflammatory signaling pathways, significantly reduces pro-inflammatory cytokine levels, and enhances macrophage differentiation into an anti-inflammatory phenotype (M2 type), thereby reducing mitochondrial ROS production and alleviating inflammation and oxidative stress. Furthermore, curcumin promotes angiogenesis and cell migration and proliferation, helping the wound surface to be quickly covered by new epithelial cells. However, the drug delivery efficiency of curcumin is limited by its poor water solubility, low bioavailability, and rapid metabolic elimination, especially its effect on biofilms formed by chronic wound infections is negligible. Therefore, direct use of curcumin cannot meet the needs of chronic wound anti-inflammatory healing.

[0068] Microneedle drug delivery is an innovative drug delivery technology that uses an array of micron-sized needles to penetrate the outermost layer of the skin, the stratum corneum, creating microchannels to deliver drugs directly to the epidermis or dermis, particularly penetrating biomembrane barriers for precise delivery to the site of infection. This technology effectively improves drug penetration efficiency and has gained widespread attention due to its painless and convenient characteristics. However, single-drug delivery using microneedles cannot achieve ideal sustained-release effects. Wound healing is a dynamic process, and precisely releasing antibacterial and anti-inflammatory drugs according to the changes in the wound healing microenvironment—on demand, at the right time, and in the right amount—remains a significant challenge.

[0069] Example 1

[0070] A multilayer microneedle patch with programmed response for sterilization and anti-inflammation, structured as follows: Figure 1 As shown, the microneedle patch has a layered structure with several needles, including an outer shell layer, a core layer and a sandwich layer. The sandwich layer wraps the core layer inside, and the outer shell layer covers the needles of the sandwich layer.

[0071] The core layer comprises polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and curcumin nanoparticles coated with ZIF-8 (zeolite imidazole acid framework-8), with a mass ratio of 1:(0.9-1.1):(1.1-1.5), preferably 1:1:1.3; PVP and PVA in the core layer are drug carriers used to load the ZIF-8 coated curcumin nanoparticles;

[0072] The outer shell layer comprises polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), and copper chloride, with a mass ratio of 1:(0.9-1.1):(0.11-0.15), preferably 1:1:0.13; PVP and PVA in the outer shell layer are drug carriers used to load the antibacterial agent copper chloride.

[0073] The sandwich layer is an enteric coating; the function of the enteric coating is pH responsive. In a specific embodiment of the present invention, the enteric coating is Eudragit S100.

[0074] The ZIF-8-coated curcumin nanoparticles have an average particle size of 120-140 nm, a drug loading of more than 10%, and an encapsulation efficiency of more than 50%.

[0075] The needles on the surface of the microneedle patch are arranged in an array. The length of a single needle is 800-900μm, the bottom diameter of the needle is 400-450μm, and the distance between the tips of two adjacent needles is 600-800μm.

[0076] The multilayer microneedle patch in this embodiment can be released in a programmed response according to changes in the wound microenvironment, initially rapidly killing bacteria and removing biofilm, and later reducing inflammation and promoting healing, thereby accelerating the healing process of chronic wound bacterial infections. The specific mechanism is as follows:

[0077] 1. Multifunctionality of the outer shell structure:

[0078] (1) Penetration mechanism: The thickness of biofilm is usually 20-200 μm, while the microneedle body is much larger than this size. At the same time, the breaking force of a single microneedle is 4.1 N, which is significantly higher than the penetration threshold of human skin. This allows the microneedle to effectively penetrate the bacterial biofilm, enabling each drug component of the microneedle to effectively act on the bacteria and the inside of the wound.

[0079] (2) Antibacterial synergy: The anaerobic fermentation process of bacteria in the biofilm of chronic wounds is accompanied by a decrease in local pH (acidic environment) and a strong inflammatory response. Under these conditions, the cell membrane integrity of some bacteria can be destroyed and biofilm formation can be inhibited.

[0080] 2. Functional design of the enteric coating Eudragit S100 sandwich structure:

[0081] (1) pH response release: The introduction of the enteric coating eliminates functional crosstalk caused by solvent erosion and component diffusion. As a large number of bacteria die, a large amount of substances are released from the cell membrane, and the pH gradually increases (neutral to alkaline environment). The ionization state of the carboxylic acid groups carried on the Eudragit S100 polymer chain changes in the neutral to alkaline environment, thereby causing changes in the polymer solubility.

[0082] (2) Harmlessness of degradation: Enteric coating Eudragit S100 is a pharmaceutical excipient listed in the pharmacopoeia. Its quality and safety have been widely recognized in clinical applications over many years, and it has excellent biocompatibility and biodegradability.

[0083] 3. Multiple functions of the core layer Cur@ZIF-8:

[0084] (1) Improved bioavailability: The Cur@ZIF-8 nanocomposite with ZIF-8 as the carrier effectively overcomes the problem of poor water solubility of free curcumin and greatly improves its bioavailability.

[0085] (2) ATP response release: ATP can compete with dimethylimidazole in the ZIF-8 structure for coordination with zinc ions;

[0086] (3) Free radical scavenging: Curcumin can efficiently scavenge reactive oxygen species (ROS) and inhibit the inflammatory cascade response triggered by oxidative stress, thereby achieving anti-oxidation and anti-inflammation;

[0087] (4) Anti-inflammatory regulation: Curcumin can reduce the expression of pro-inflammatory factors (TNF-α, IL-6) in cells by regulating macrophage polarization (promoting the M2 anti-inflammatory phenotype);

[0088] (5) Promotes healing: The released curcumin has the properties of promoting angiogenesis and cell migration, which can promote wound healing.

[0089] 4. Spatiotemporal synergistic mechanism:

[0090] (1) Cascade reaction regulation: In the early stage of the release of copper ions in the shell, they can be delivered to the deep infection to destroy the bacterial biofilm. As a large number of bacteria die, the pH and ATP content gradually increase, which promotes the swelling of the enteric coating, the disintegration of the ZIF-8 structure, and the continuous release of curcumin, which eliminates excess ROS, promotes angiogenesis and cell migration, and accelerates wound healing.

[0091] (2) Intelligent dual-response design: The synergy of pH response and ATP response can achieve precise treatment of chronic wound infection.

[0092] In summary, the multi-layer microneedle patch provided by this invention, through a multimodal synergistic strategy of curcumin nano-coated particles and enteric coating, overcomes the limitation of traditional antibacterial materials that cannot simultaneously achieve anti-inflammatory functions, providing a new approach for the comprehensive treatment of infectious inflammation, and can accelerate the healing of bacterial infectious chronic wounds.

[0093] Example 2

[0094] Preparation method of ZIF-8 coated curcumin nanoparticles:

[0095] First, dissolve 300 mg of zinc nitrate hexahydrate in 10 mL of water to prepare solution A. Then, disperse 657 mg of 2-methylimidazole and 20 mg of curcumin in 20 mL of methanol solution in sequence, allowing the hydrophobic curcumin molecules and 2-methylimidazole ligands to undergo molecular-level interaction in methanol to form a homogeneous mixed precursor liquid B.

[0096] The two liquids were rapidly mixed and sonicated for 2 minutes, then stirred at 600 rpm for 15 minutes. After the reaction was complete, the material was washed with methanol until the supernatant was nearly colorless and transparent. After drying, Cur@ZIF-8 nanoparticles were obtained.

[0097] Through experimental verification, the inventors found that when the mass ratio of zinc nitrate hexahydrate, 2-methylimidazole, and curcumin was 30:65.7:2, the ZIF drug loading was 15.0% and the encapsulation efficiency was 73.4%. When the mass of curcumin was reduced to a ratio of 30:65.7:1, the ZIF drug loading was 10.5% and the encapsulation efficiency was 85.7%. When the mass of curcumin was increased to a ratio of 30:65.7:3, the ZIF drug loading was 10.2% and the encapsulation efficiency was 57.3%. Therefore, considering both drug loading and encapsulation efficiency, this invention selects a mass ratio of zinc nitrate hexahydrate, 2-methylimidazole, and curcumin of 30:65.7:2 to prepare Cur@ZIF-8 nanoparticles, ensuring both high drug loading and encapsulation efficiency.

[0098] Example 3

[0099] A method for preparing a programmed response antibacterial and anti-inflammatory multilayer microneedle patch, the process of which follows the procedure... Figure 2 The procedure is as shown and includes the following steps:

[0100] (1) Add 3.75 g PVP to 50 ml of deionized water at a constant temperature of 65℃, disperse evenly, and then slowly add 3.75 g PVA and stir overnight. When using, add 5 g Cur@ZIF-8 nanoparticles and ultrasonically disperse as the core layer microneedle precursor solution.

[0101] (2) Add 300 μL of core layer microneedle precursor solution to the microneedle mold, centrifuge at 6000 rpm for 5 min to fully fill and remove air bubbles, and then place it in a constant temperature drying oven to dry for 12 h. After drying, it is taken out for use, and the microneedle core layer Cur@ZIF-8-MN (CZ-MN) with anti-inflammatory function is obtained.

[0102] (3) Add 2.5 g Eudragit S100 to 50 mL of ethanol at a constant temperature of 65℃, stir and sonicate to disperse evenly as a precursor solution for sandwich microneedles.

[0103] (4) Add 150 μL of sandwich microneedle precursor solution to the microneedle mold, centrifuge at 6000 rpm for 5 min and pre-dry for 1 h. Use the microneedle core layer obtained in step (2) as a positive mold and press it into the mold filled with sandwich microneedle precursor solution. After drying for 12 h, add 150 μL of sandwich microneedle precursor solution for encapsulation. After final drying, take it out to obtain double-layer microneedles CZ@EC-MN (CZ@E-MN) with enteric coating protection and pH response characteristics.

[0104] (5) Add 3.75 g PVP to 50 ml of deionized water at a constant temperature of 65℃, mix well, add 0.5 g CuCl2, dissolve completely, and then slowly add 3.75 g PVA and stir overnight to prepare the microneedle precursor solution for the outer shell layer.

[0105] (6) Add the outer shell layer microneedle precursor liquid to the microneedle mold, centrifuge and dry, use the double-layer microneedle CZ@EC-MN obtained in step (4) as a positive mold, press it into the mold filled with the outer shell layer microneedle precursor liquid, dry, and obtain multilayer microneedle patch CZ@EC@Cu-MN (CZ@E@C-MN).

[0106] Experimental Example

[0107] The Cur@ZIF-8 nanoparticles prepared in Example 2 and the multilayer microneedle patch CZ@EC@Cu-MN prepared in Example 3 were subjected to relevant experimental verification, specifically including the following contents.

[0108] 1. Morphology experiments of Cur@ZIF-8 and CZ@E@C-MN multilayer microneedles

[0109] (1) SEM characterization: The morphology of the prepared Cur@ZIF-8 nanoparticles and CZ@E@C-MN multilayers was characterized by SEM, and the results are as follows: Figure 3 and Figure 4 As shown.

[0110] (2) Fluorescence detection: To verify the multilayer structure distribution of the microneedles, the shell and core layers of the CZ@E@C-MN multilayer microneedles were stained with isothiocyanate dye FITC (green) and carbocyanine dye Dil (red), respectively, thus producing green and red fluorescence. Cross-sectional images of the CZ@E@C-MN multilayer microneedles were captured using a laser confocal microscope, and the results are as follows: Figure 5 As shown, the results confirm the existence of the multilayer structure.

[0111] (3) Particle size analysis: Statistical analysis was performed on the particle size of the prepared Cur@ZIF-8 nanoparticles. The results are as follows: Figure 6 As shown, the average particle size of the curcumin nanoparticles coated with ZIF-8 is 133±4 nm.

[0112] 2. Performance testing of CZ@E@C-MN multilayer microneedles

[0113] (1) Mechanical property testing of CZ@E@C-MN multilayer microneedles: Fix the microneedle array sample on the sample stage of the texture analyzer, ensuring that the surface of the microneedle array is flat and perpendicular to the probe; turn on the texture analyzer, complete the calibration and set it to compression mode, adjust the relevant test parameters, align the probe with the center of the microneedle array, start the test program, stop the test when the preset distance is reached or the microneedle breaks, record the force-displacement curve, and the results are as follows. Figure 7 As shown, the mechanical strength of CZ@E@C-MN multilayer microneedles is approximately 4.1 N / needle.

[0114] (2) POD-like activity test of CZ@E@C-MN multilayer microneedles: CZ@E@C-MN multilayer microneedle patches were immersed in PBS at pH 5.5, and the immersion solutions were collected at different times (5, 10, 15 min). The solutions were added to 2 mL of PBS containing 1 mM TMB and 5 mM H2O2. After reacting for 1 min, the absorbance curve was measured to obtain the POD-like activity of the CZ@E@C-MN multilayer microneedle patch immersion solution as shown in the figure. Figure 8 As shown.

[0115] Among them, such as Figure 8 As shown in Figure A, microneedles exhibit excellent POD-like activity under acidic conditions. To investigate the source of the POD-like activity of the CZ@E@C-MN multilayer microneedle patch, the POD-like activity of different components of the CZ@E@C-MN multilayer microneedle patch was measured, and the results are as follows. Figure 8 As shown in B, the POD-like activity originates from the shell copper ions, and the results of the CZ@C-MN group without the enteric coating are similar to those of Cu. 2+ The results for the +Cur group were similar, indicating that without the protection of the enteric coating, the premature release of curcumin would greatly reduce the POD-like activity of the microneedle patch.

[0116] (3) Dual-response release of curcumin from CZ@E@C-MN multilayer microneedles: The microneedles were immersed in 2 mL of PBS with different pH and ATP concentrations. The release rate of curcumin was determined at regular intervals using the characteristic absorption of curcumin at 425 nm. The results are as follows: Figure 9 As shown, the material only exhibits sustained curcumin release in the presence of ATP at a neutral to alkaline pH.

[0117] 3. Antibacterial performance test of CZ@E@C-MN multilayer microneedles

[0118] The plate count method was used to detect the effectiveness of CZ@E@C-MN multilayer microneedles in killing Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli). Under simulated endogenous hydrogen peroxide concentration conditions (10 μM), each group was mixed with bacterial suspension to obtain a mixed treatment solution, which was incubated for 3 h. The results are as follows: Figure 10 As shown.

[0119] Compared to the control group, the CZ@C-MN group showed a weaker antibacterial effect. This is because the multilayer microneedles without an enteric coating experienced functional crosstalk, leading to premature release of curcumin. This premature release cleared a large amount of reactive oxygen species before the bacteria died, significantly reducing the antibacterial effect. However, the number of bacteria co-incubated with the CZ@E@C-MN multilayer microneedles was extremely small, indicating that the CZ@E@C-MN multilayer microneedles have a good bactericidal effect and highlighting the necessity of the time-sequential release of these microneedles.

[0120] 4. Biofilm clearance performance test of CZ@E@C-MN multilayer microneedles

[0121] Staphylococcus aureus (10) was used in 24-well plates 7 The effect of CZ@E@C-MN multilayer microbes on biofilm removal was evaluated by culturing in trypsin-soybean broth (TSB) medium (CFU / mL) for 48 h to form a dense biofilm. Each group of microbes was used to treat the biofilm. The remaining biofilm at the bottom of the 24-well plates was fixed with 4% paraformaldehyde and stained with crystal violet for 10 min. Residual dye was washed with 30% acetic acid, and the eluent was measured at 590 nm using an ELISA reader. The results are shown below. Figure 11 As shown.

[0122] Depend on Figure 11 The results are consistent with the trend of the antibacterial performance test of Z@E@C-MN multilayer microneedles. The absorbance of the biofilm in the CZ@E@C-MN multilayer microneedle group was significantly reduced, which further demonstrates the good biofilm removal effect of CZ@E@C-MN multilayer microneedles and the necessity of time-sequential release.

[0123] 5. Biocompatibility test of CZ@E@C-MN multilayer microneedles

[0124] HUVEC cells were co-cultured with CZ@E@C-MN multilayer microneedle soaking solution for 12-24 hours. Cell viability was assessed using CCK-8 assay to evaluate the cell compatibility of CZ@E@C-MN multilayer microneedles. After 24 hours of co-incubation with CZ@E@C-MN multilayer microneedle soaking solution, cell viability was determined using fluorescence staining. The results are as follows: Figure 12 As shown.

[0125] Depend on Figure 12It was found that the cells in the CZ@E@C-MN multilayer microneedle group maintained cell viability comparable to the control group, indicating that the CZ@E@C-MN multilayer microneedles have almost no toxicity to cells, and most HUVEC cells were in a healthy growth state (green indicates live cells), with almost no dead cells (red indicates dead cells). These results demonstrate that the CZ@E@C-MN multilayer microneedles have good cell compatibility.

[0126] 6. Anti-inflammatory performance test of CZ@E@C-MN multilayer microneedles

[0127] ROS was detected using the DCFH-DA reagent, a ROS fluorescent probe. Cells treated with 300 μM H2O2 alone served as a positive control. The experimental group consisted of cells incubated with a mixture of 300 μM H2O2 and CZ@E@C-MN multilayer microneedle soaking solution, followed by fluorescent staining with DCFH-DA and Hoechst 33342. The results are shown below. Figure 13 As shown.

[0128] Figure 13 The fluorescence in the cells is shown, where green fluorescence comes from DCFH-DA, indicating that the reagent has bound to ROS, while blue fluorescence comes from Hoechst 33342, representing the cell nucleus; Figure 13 It can be seen that the green fluorescence intensity of CZ@E@C-MN multilayer microneedles is significantly lower than that of the H2O2 positive control, proving that CZ@E@C-MN multilayer microneedles have good anti-inflammatory effects.

[0129] 7. Cell migration promotion performance assay of CZ@E@C-MN multilayer microneedles

[0130] The cell migration-promoting effect of CZ@E@C-MN multilayer microneedles was assessed using a cell scratch assay on HUVEC cells. HUVEC cells were seeded in 24-well plates at a cell density of 5 × 10⁶ cells / well. 5 Cells were cultured in complete medium containing 10% fetal bovine serum (FBS) per well. After 16-24 hours of culture, a monolayer of cells formed. A straight line was drawn using the tip of a 200 μL pipette. Cell debris was removed by washing with sterile PBS, and then sterile CZ@E@C-MN multilayer microneedle soaking solution was added to the wells. A well containing complete medium served as a control group. Cells were cultured at 37°C and 5% CO2 for 48 hours. Finally, the medium, materials, and cell debris were removed. The cell scratch was photographed using an inverted microscope. The results are shown below. Figure 14 As shown.

[0131] Depend on Figure 14It can be seen that after 24 h of culture, the cells migrated towards the central scratch. Compared with the control group, the scratches on the CZ@E@C-MN multilayer microneedles almost completely disappeared, indicating that the CZ@E@C-MN multilayer microneedles have the ability to promote cell migration.

[0132] 8. Wound healing performance test of CZ@E@C-MN multilayer microneedles

[0133] A diabetic mouse model was established. C57 mice were selected and randomly divided into two groups of six each. Mice were acclimatized to the environment for one week prior to modeling. Mice were fasted for 8 hours before modeling and injected intraperitoneally daily with streptozotocin (STZ, dissolved in sodium citrate buffer at pH 4.5) at a dose of 50 mg / kg body weight. A high-sugar, high-fat diet was used during modeling. Modeling was considered successful when the random / satiety blood glucose level was ≥16.7 mM.

[0134] Hair was removed from diabetic model mice, and a full-thickness wound of approximately 10 mm was created on the skin of their backs. Staphylococcus aureus (at a concentration of 10) was then inoculated into the full-thickness wound. 8 CFU / mL) for 2 days to allow for the completion of chronic wound formation in diabetic mice. CZ@E@C-MN multilayer microneedles were administered to the wounds of mice every other day, and wound healing and histological analysis were observed. The results are as follows: Figure 15 , Figure 16 and Figure 17 As shown. Among them, Figure 15 The image shows the wound and its condition in the mouse. Figure 16 HE-stained images of mouse wound skin 14 days after treatment are shown. Figure 17 The image shows Masson staining of mouse wound skin.

[0135] Depend on Figure 15 It can be seen that as the treatment time of CZ@E@C-MN multilayer microneedling increases, the wound area in mice treated with CZ@E@C-MN multilayer microneedling gradually decreases, and the healing at each time point is significantly better than that in the control group. From Figure 15 It can be seen that the wound healing rate of mice is relatively fast. After 14 days, the CZ@E@C-MN multilayer microneedle group has basically been completely healed, which shows that mice have a good ability to promote wound healing.

[0136] HE staining was performed on the wound tissue to analyze histological changes at the wound site. Figure 16It can be seen that the control group showed no obvious signs of healing and the lesions were severe, with a small amount of parakeratosis, inflammatory cell infiltration, fibroblast proliferation, and a large amount of granular layer thickening. In contrast, the CZ@E@C-MN multilayer microneedling showed significant improvement, with a large amount of hyperkeratosis, a small amount of granular layer thickening and inflammatory cell infiltration, and a small number of new hair follicles. That is, the tissue remodeling was almost completed, and there were almost no inflammatory cells. It was characterized by an epidermal thickness close to that of normal skin, a narrow dermal gap, a relatively normal dermal layer, and a large number of hair follicle structures.

[0137] The collagen content in wound tissue was analyzed using Masson's trichrome staining method, and the results are as follows: Figure 17 As shown in the figure, in the control group, only a small amount of collagen deposition was observed, while in the CZ@E@C-MN multilayer microneedles, collagen fibers were observed to exhibit a more orderly bundled arrangement (blue), and there was obvious collagen fiber deposition in the granulation tissue. The collagen distribution was relatively uniform, indicating that the wound healing process was basically completed.

[0138] In summary, this invention provides a multilayer microneedle patch with programmed response for sterilization and anti-inflammation. Through structural synergy and component design, it can simultaneously enhance the sterilization and anti-inflammatory effects of multilayer microneedles. In actual experiments, this multilayer microneedle exhibited good antibacterial, anti-inflammatory, and wound-healing capabilities. The multilayer microneedle achieves its antibacterial, anti-inflammatory, and wound-healing-promoting processes through rapid sterilization of the shell layer and the sequential release of curcumin in a dual-response manner, demonstrating the application potential of multilayer microneedles as a biomedical material.

[0139] Furthermore, the preparation method of the present invention only requires a synthesis and coating process under mild conditions. The overall preparation method is simple and easy to operate, and can be applied on a large scale in industry.

[0140] Based on this, the present invention also provides a wound healing material, which is based on the antibacterial, anti-inflammatory and wound healing promoting effects of multilayer microneedles, and can provide a new biomaterial solution for the treatment of diabetic chronic wounds that integrates efficient antibacterial, inflammation regulation and tissue regeneration.

[0141] Finally, it should be noted that the above specific embodiments are only used to describe the purpose, technical solution, and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation of the present invention and is not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the foregoing specific embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions or improvements can be made to some or all of the technical features. These modifications, equivalent substitutions, and improvements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A bactericidal and anti-inflammatory multi-layer microneedle patch with programmed response, characterized in that, The microneedle patch has a layered structure with several needles, including an outer shell layer, a core layer and a sandwich layer. The sandwich layer wraps the core layer inside, and the outer shell layer covers the needles of the sandwich layer. The core layer comprises polyvinylpyrrolidone, polyvinyl alcohol, and ZIF-8-coated curcumin nanoparticles, with a mass ratio of 1:(0.9-1.1):(1.1-1.5). The outer shell consists of polyvinylpyrrolidone, polyvinyl alcohol, and copper chloride, in a mass ratio of 1:(0.9-1.1):(0.11-0.15). The sandwich layer is an enteric coating Eudragit S100.

2. The multi-layered microneedle patch with programmed response according to claim 1, wherein, The ZIF-8-coated curcumin nanoparticles have an average particle size of 120-140 nm, a drug loading of more than 10%, and an encapsulation efficiency of more than 50%.

3. The multi-layered microneedle patch with programmed response according to claim 1, wherein, The needles on the surface of the microneedle patch are arranged in an array.

4. The multi-layered microneedle patch with programmed response according to claim 1, wherein, The length of a single needle is 800-900μm, the diameter of the bottom of the needle is 400-450μm, and the distance between the tips of two adjacent needles is 600-800μm.

5. The method of claim 1-4 for the preparation of the bactericidal and anti-inflammatory multi-layer microneedle patch with programmed response, characterized in that, Includes the following steps: Add the core layer microneedle precursor solution to the microneedle mold, centrifuge and dry to obtain the microneedle core layer Cur@ZIF-8-MN; Add the sandwich layer microneedle precursor liquid to the microneedle mold, centrifuge and dry. Use the microneedle core layer Cur@ZIF-8-MN obtained in the previous step as a positive mold, press it into the mold filled with the sandwich layer microneedle precursor liquid, dry it, add the sandwich layer microneedle precursor liquid for encapsulation, and dry it to obtain the enteric-coated double-layer microneedles CZ@EC-MN. Add the outer shell layer microneedle precursor liquid to the microneedle mold, centrifuge and dry. Use the bilayer microneedle CZ@EC-MN obtained in the previous step as a positive mold, press it into the mold filled with the outer shell layer microneedle precursor liquid, and dry to obtain the multilayer microneedle patch CZ@EC@Cu-MN.

6. The method for preparing a programmed response bactericidal and anti-inflammatory multilayer microneedle patch according to claim 5, characterized in that, The preparation method of the core layer microneedle precursor fluid is as follows: Polyvinylpyrrolidone was added to deionized water in a certain proportion and dispersed evenly. Polyvinyl alcohol was then slowly added and stirred. ZIF-8-coated curcumin nanoparticles were added and ultrasonically dispersed to obtain the core layer microneedle precursor fluid.

7. The method of claim 6, wherein the method further comprises the step of: The method for preparing the ZIF-8-coated curcumin nanoparticles is as follows: ​ Zinc nitrate hexahydrate was dissolved in water to prepare an aqueous solution of zinc nitrate. 2-methylimidazole and curcumin were then dispersed sequentially into a methanol solution to form a homogeneous mixed precursor liquid. The two liquids were rapidly mixed, subjected to ultrasonic and stirring treatment, and after the reaction was completed, they were washed and dried to obtain Cur@ZIF-8 nanoparticles. The mass ratio of zinc nitrate hexahydrate, 2-methylimidazole, and curcumin is 30:(60-70):(1-3).

8. The method for preparing a programmed response bactericidal and anti-inflammatory multilayer microneedle patch according to claim 5, characterized in that, The preparation method of the sandwich layer microneedle precursor fluid is as follows: Enteric coating was added to ethanol, and the mixture was stirred and ultrasonically dispersed to obtain a sandwich microneedle precursor solution.

9. The method for preparing a programmed response bactericidal and anti-inflammatory multilayer microneedle patch according to claim 5, characterized in that, The preparation method of the outer shell microneedle precursor fluid is as follows: Polyvinylpyrrolidone was added to deionized water in a certain proportion, mixed well, and then copper chloride was added to dissolve it completely. Polyvinyl alcohol was then slowly added and stirred evenly to obtain the microneedle precursor solution for the outer shell layer.

10. A wound healing material, characterized in that, It includes a multilayer microneedle patch with a programmed response as described in any one of claims 1-4.

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