Multifunctional microneedle with core-shell structure and preparation method and application thereof

Abstract

The application discloses a multifunctional microneedle with a core-shell structure and a preparation method and application thereof, and relates to the technical field of biomedical materials.The multifunctional microneedle takes a methacrylic anhydride modified biomacromolecule material and spermidine as a needle tip material, fills the methacrylic anhydride modified biomacromolecule material and spermidine into micropores of a microneedle mold by a centrifugal method, and constructs a microneedle core layer by free radical polymerization of the methacrylic anhydride modified biomacromolecule material under light initiation; then, a mixed solution of a polymer base material and metal-polyphenol nanomaterial is injected into the microneedle mold, and the microneedle mold is dried and demolded, so that the multifunctional microneedle with the core-shell structure is obtained.The multifunctional microneedle with the core-shell structure can promote the release of effective components in a dermis layer of skin, has multiple functions such as antibiosis and promotion of blood vessel regeneration, and can realize high-quality healing of a wound surface.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a multifunctional microneedle with a core-shell structure, its preparation method, and its application. Background Technology

[0002] Diabetes mellitus is one of the top ten causes of death worldwide. Its core pathological feature is chronic hyperglycemia, often accompanied by various complications such as nephropathy, retinopathy, peripheral neuropathy, and chronic wounds. According to the International Diabetes Federation, there are currently over 500 million people with diabetes globally, and this number is projected to reach 850 million adults by 2050, placing a significant burden on global healthcare and the economy. Among the many complications, diabetic chronic wounds are considered one of the most serious due to their high incidence and difficulty in healing. As the duration of the disease increases, these wounds not only severely impact patients' physical and mental health and quality of life but also significantly increase the risk of disability, amputation, and even death.

[0003] When normal skin is damaged, the body sequentially goes through four stages—hemostasis, inflammation, proliferation, and remodeling—to achieve wound self-healing. However, under abnormal microenvironmental stimuli such as high glucose and oxidative stress, diabetic chronic wounds often remain stuck in the inflammatory phase. This is mainly due to the continuous activation of pro-inflammatory macrophages (M1 type), which release large amounts of pro-inflammatory factors, such as interleukin-6, tumor necrosis factor-α, and interleukin-1β. The overexpression of these pro-inflammatory factors, on the one hand, recruits more inflammatory cells to the wound, further releasing inflammatory mediators and amplifying the inflammatory response; on the other hand, they also promote the expression of matrix metalloproteinases while inhibiting the production of their inhibitors, disrupting the balance between extracellular matrix degradation and generation, thus preventing diabetic chronic wounds from entering the proliferation phase. Because chronic wounds remain in the inflammatory phase for a long time and cannot close effectively, patients face a higher risk of microbial and pathogenic infection. Once infection occurs, it will not only exacerbate the local inflammatory response of the wound but may also trigger systemic infection, which can be life-threatening in severe cases.

[0004] Therefore, there is an urgent need to develop an integrated material that can break through tissue barriers, achieve deep delivery, and synergistically regulate inflammation, eliminate reactive oxygen species and pathogens. Summary of the Invention

[0005] To address the challenges of reactive oxygen species (ROS) accumulation, pathogen infection, persistent inflammatory response, and impaired angiogenesis in diabetic chronic wounds, this invention provides a multifunctional microneedle with a core-shell structure, its preparation method, and its application. Through structural design and functional integration, this multifunctional microneedle can achieve multiple synergistic effects, including ROS scavenging, antibacterial activity, immune regulation, and promotion of angiogenesis, providing a new strategy for promoting the healing of diabetic chronic wounds.

[0006] According to one aspect of the present invention, a multifunctional microneedle with a core-shell structure is provided. The microneedle tip material is a methacrylic anhydride-modified biopolymer and spermidine, which are filled into the micropores of a microneedle mold using a centrifugal method. The core layer of the microneedle is then constructed by photo-initiated free radical polymerization of the methacrylic anhydride-modified biopolymer. Next, a mixture of a polymer base material and a metal-polyphenol nanomaterial is injected into the microneedle mold, and the mold is dried and demolded to obtain the multifunctional microneedle with a core-shell structure.

[0007] Optionally, the methacrylic anhydride-modified biopolymer material is any one of methacrylamide hyaluronic acid, methacrylamide gelatin, methacrylamide collagen, methacrylamide carboxymethyl chitosan, and methacrylamide sodium alginate.

[0008] Optionally, the metal-polyphenol nanomaterial is any one of copper-tannic acid nanoparticles, iron-tannic acid nanoparticles, silver-curcumin nanoparticles, copper-epigallocatechin gallate nanoparticles, and iron-gallic acid nanoparticles.

[0009] Optionally, the polymer substrate material is any one of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, polylactic acid, and polylactic acid-polyglycolic acid copolymer.

[0010] Optionally, the photoinitiator used for photoinitiation is either lithium phenyl (2,4,6-trimethylbenzoyl)phosphate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.

[0011] According to another aspect of the present invention, a method for preparing a multifunctional microneedle with a core-shell structure is provided, comprising: S1, Preparation of methacrylic anhydride-modified biopolymer materials: Biopolymer materials are dissolved in deionized water, and methacrylic anhydride is added dropwise to cause the methacrylic anhydride to undergo an acylation reaction with the amino or hydroxyl groups in the biopolymer materials. At the same time, sodium hydroxide solution is used to adjust the pH of the reaction solution. After the reaction is complete, impurities are removed by dialysis, and the biopolymer materials modified with methacrylic anhydride are obtained by freeze drying. S2, Preparation of metal-polyphenol nanomaterials: Metal ions and polyphenols were dissolved in deionized water, sodium hydroxide solution was added to adjust the pH of the reaction system, the mixture was stirred in a water bath, the precipitate was collected by centrifugation and washed with deionized water and ethanol, and the product was vacuum dried to obtain metal-polyphenol nanomaterials. S3, Preparation of needle tip solution: The photoinitiator was dissolved in PBS buffer and stirred in the dark to promote complete dissolution. Then, methacrylic anhydride-modified biopolymer and spermidine solution were added to obtain the needle tip solution. S4, Preparation of the base solution: Metal-polyphenol nanomaterials were dispersed in PBS buffer, followed by the addition of polymer substrate material. The mixture was stirred until it was completely dissolved to obtain the substrate solution. S5, Preparation of multifunctional microneedles with core-shell structure: The tip liquid was injected into the microneedle mold, centrifuged to remove air bubbles and fill the micropores of the mold, and the mold was placed in a forced-air drying oven for concentration. The concentration step was repeated several times. Then, the tip liquid was irradiated with ultraviolet light to initiate free radical polymerization of the methacrylic anhydride modified biopolymer material to form a cross-linked network. Finally, the base liquid was added to the mold, and after drying and demolding, a multifunctional microneedle with a core-shell structure was obtained.

[0012] Optionally, the reaction temperature in S1 is 4℃ ~ 50℃, and the reaction time is 2 h ~ 24 h; the reaction process in S2 is carried out at 50℃ under light-protected conditions; and the ultraviolet irradiation time in S5 is 5 s ~ 60 s.

[0013] Optionally, in the needle tip solution obtained in S3, the concentration of the photoinitiator is 0.05 wt% ~ 0.3 wt%, the concentration of the methacrylic anhydride modified biopolymer is 5 wt% ~ 15 wt%, and the concentration of spermidine is 1.0 mM ~ 10 mM.

[0014] Optionally, in the substrate solution obtained in S4, the concentration of metal-polyphenol nanomaterials is 0.1 mg / mL to 0.5 mg / mL, and the concentration of polymer substrate material is 5 wt% to 40 wt%.

[0015] According to another aspect of the present invention, the application of a multifunctional microneedle with a core-shell structure as described above, or a multifunctional microneedle with a core-shell structure prepared by the preparation method described above, in the preparation of a material for healing chronic wounds in diabetic patients is provided.

[0016] The technical solution proposed in this application has the following beneficial effects: a) Based on their unique morphology and geometry, microneedles can painlessly puncture skin barriers, promoting the release of active ingredients in the dermis and achieving high-quality healing of chronic diabetic wounds. Furthermore, the use of biopolymer materials to construct microneedles further enhances the therapeutic effect. On the one hand, biopolymer materials have excellent biocompatibility, avoiding exacerbation of inflammatory responses in chronic wounds; on the other hand, the small molecules produced during their degradation provide nutrients for processes such as cell proliferation, collagen deposition, and skin appendage regeneration, further accelerating the healing of chronic wounds.

[0017] (b) Microneedles with a core-shell structure were constructed using different cross-linking methods to achieve sequential drug delivery. The core layer of the microneedle undergoes photo-initiated free radical polymerization of methacrylamide biopolymers, forming a dense and stable covalently cross-linked network for loading spermidine. The shell layer is composed of a polymer substrate material and metal-polyphenol nanomaterials through physical cross-linking methods such as hydrogen bonding and electrostatic interactions. When the microneedles come into contact with the tissue fluid of a chronic diabetic wound, the shell layer rapidly swells and may even dissociate, thereby rapidly releasing the loaded metal-polyphenol nanomaterials for rapid, explosive drug delivery. As the shell layer degrades, the core layer structure is exposed to the wound microenvironment and slowly degrades under the synergistic action of various enzymes, thus achieving a slow and sustained release of spermidine.

[0018] c) Loading metal-polyphenol nanomaterials onto microneedles allows for effective disruption of bacterial biofilm structures through the physical penetration properties of the microneedles, thereby precisely delivering the metal-polyphenol nanomaterials to deep within infected tissues. Stimulated by the microenvironment of diabetic chronic wounds, the microneedles release metal ions and polyphenols. These ions work synergistically through multiple mechanisms, including disrupting bacterial cell structure and function and inducing oxidative stress, ultimately achieving highly efficient clearance of pathogens.

[0019] d) Co-loading metal-polyphenol nanomaterials with spermidine into microneedles enables multi-faceted immunomodulation of diabetic chronic wounds. Under the stimulation of the microenvironment of the diabetic chronic wound, the microneedles exhibit a cascade effect: the preferentially released metal-polyphenol nanomaterials, with their diverse enzyme activities, effectively scavenge excess reactive oxygen species in the wound, alleviating oxidative stress damage. Subsequently released polyphenols and spermidine synergistically regulate macrophage polarization, promoting the transformation of macrophages from pro-inflammatory macrophages (M1 type) to anti-inflammatory macrophages (M2 type), thereby accelerating the resolution of the inflammatory phase of diabetic chronic wounds and propelling the healing process towards the proliferative phase. Attached Figure Description

[0020] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the preparation mechanism of the multifunctional microneedles with a core-shell structure in an embodiment of the present invention, wherein A is the step of preparing methacrylic anhydride modified hyaluronic acid, B is the step of preparing copper-tannic acid nanoparticles, and C is the step of preparing multifunctional microneedles with a core-shell structure. Figure 2 These are bright-field images and corresponding inverted fluorescence images of dual-dye labeled microneedles under different views, representing embodiments of the present invention. Figure 3 The cytotoxicity of multifunctional microneedles with a core-shell structure is demonstrated in this embodiment of the invention. Figure 4The antibacterial properties of multifunctional microneedles with a core-shell structure are illustrated in the embodiments of the present invention. Figure 5 This is a histological analysis of a multifunctional microneedle with a core-shell structure used in the treatment of chronic diabetic wounds, as described in an embodiment of the present invention. Detailed Implementation

[0021] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all of them. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

[0022] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0023] Unless otherwise specified, all conditions in the examples were performed under standard conditions or according to the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are commercially available standard products. Unless otherwise stated, all technical and scientific terms herein have the meanings commonly understood by one of ordinary skill in the art. Example

[0024] This embodiment provides a stepwise method for preparing multifunctional microneedles with a core-shell structure: First, methacrylic anhydride-modified biopolymer and metal-polyphenol nanomaterials are prepared separately; then, a mixture of a photoinitiator, methacrylic anhydride-modified biopolymer, and spermidine is prepared to obtain a tip solution, and a mixture of metal-polyphenol nanomaterials and a polymer substrate is prepared to obtain a substrate solution; finally, multifunctional microneedles with a core-shell structure are prepared by a template method.

[0025] Among them, the methacrylic anhydride modified biopolymer material includes at least one of methacrylamide hyaluronic acid (HAMA), methacrylamide gelatin (GelMA), methacrylamide collagen (CoIMA), methacrylamide carboxymethyl chitosan (CMSA), and methacrylamide sodium alginate (SAMA).

[0026] Metal-polyphenol nanomaterials include at least one of copper-tannic acid nanoparticles (CuTA NPs), iron-tannic acid nanoparticles (FeTA NPs), silver-curcumin nanoparticles (Ag-Cur NPs), copper-epigallocatechin gallate nanoparticles (Cu-EGCG NPs), and iron-gallic acid nanoparticles (Fe-GA NPs).

[0027] The polymer substrate material includes at least one of hyaluronic acid (HA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polylactic acid (PLA), and polylactic acid-polyglycolic acid copolymer (PLGA).

[0028] The photoinitiator is either lithium phenyl (2,4,6-trimethylbenzoyl)phosphate (LAP) or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone (I2959).

[0029] The preparation method of this multifunctional microneedle with a core-shell structure includes the following steps: a) Preparation of methacrylic anhydride-modified biopolymers Under light-protected conditions at 4℃ to 50℃, the biopolymer material was dissolved in deionized water with continuous stirring until completely dissolved. Then, methacrylic anhydride was added dropwise to the reaction solution, and the pH was adjusted with sodium hydroxide solution to control the reaction progress. After the reaction was complete (approximately 2 h to 24 h), the reaction system was transferred to a dialysis bag to remove raw materials, byproducts, and impurities. The dialysis product was collected and freeze-dried to obtain the methacrylic anhydride-modified biopolymer material.

[0030] b) Preparation of metal-polyphenol nanomaterials Metal ion salts and polyphenolic compounds are dissolved in deionized water. After adjusting the pH of the reaction solution, the mixture is continuously stirred to promote a chelation reaction between the metal ions and the phenolic hydroxyl groups in the polyphenol structure (preferably, the reaction conditions are 50°C in the dark), forming nanomaterials. After the reaction is complete, unreacted raw materials and byproducts are removed by centrifugation, and the precipitate is dried under vacuum at 60°C to obtain the metal-polyphenol nanomaterials.

[0031] c) Preparation of needle tip solution Under conditions of 50°C and protection from light, the photoinitiator powder was dissolved in PBS buffer at pH = 7.4. Then, the methacrylic anhydride-modified biopolymer material solid and spermidine solution prepared in step a were added sequentially and stirred until completely dissolved to obtain the needle tip solution.

[0032] d) Preparation of base solution Under room temperature and light-protected conditions, the metal-polyphenol nanomaterials prepared in step b were uniformly dispersed in PBS buffer at pH = 7.4, and then the polymer substrate material was added. After stirring evenly, the substrate solution was obtained.

[0033] e) Fabrication of multifunctional microneedles with core-shell structures The tip solution prepared in step c is injected into the microneedle mold, and centrifugation is used to ensure it fully fills the micropores of the mold. Multiple concentration steps are then performed to increase the loading of the active ingredients in the tip solution. Subsequently, the tip solution is irradiated with ultraviolet light for 5 to 60 seconds to induce cross-linking and curing. Next, the base solution prepared in step d is injected into the mold, and after drying and demolding, a multifunctional microneedle with a core-shell structure is obtained.

[0034] In step c, the concentration of the photoinitiator in the needle tip solution is 0.05 wt% ~ 0.3 wt%, the concentration of the methacrylic anhydride modified biopolymer is 5 wt% ~ 15 wt%, and the concentration of spermidine is 1.0 mM ~ 10 mM; in step d, the concentration of the metal-polyphenol nanomaterial is 0.1 mg / mL ~ 0.5 mg / mL, and the concentration of the polymer base material is 5 wt% ~ 40 wt%. Example

[0035] Compared to Example 1, this example provides specific materials and operating conditions for preparing multifunctional microneedles with a core-shell structure. For example... Figure 1 As shown, in this embodiment, the specific preparation steps are as follows: a) Preparation of methacrylic anhydride-modified hyaluronic acid 6 g of hyaluronic acid (HA) was weighed and placed in a 500 mL reactor. 300 mL of deionized water was added, and the mixture was stirred at 4°C in the dark for 4 h to ensure complete dissolution. Then, 11.7 mL of methacrylic anhydride was added dropwise to the hyaluronic acid solution using a 5.0 mL dropper. During the reaction, the pH of the reaction solution was controlled at 8–10 using a 5 M sodium hydroxide aqueous solution. After reacting in the dark for 24 h, the reaction system was transferred to a dialysis bag with a molecular weight cutoff of 8000 Da–14000 Da and dialyzed against deionized water for 5 days to remove unreacted methacrylic anhydride and byproducts. The dialysate was freeze-dried to obtain a white, cotton-like solid, namely methacryloyl hyaluronic acid (HAMA).

[0036] b) Preparation of copper-tannic acid nanoparticles 3.5 g of copper sulfate pentahydrate (CuSO4·5H2O) and 104 mg of tannic acid (TA) were weighed and dissolved in 200 mL of H2O. The solution was then sonicated to promote complete dissolution. At room temperature, the pH of the reaction solution was adjusted to 7.4 using an aqueous sodium hydroxide solution. The reaction system was then transferred to a 50°C water bath and stirred continuously for 2–3 h. The precipitate was collected by centrifugation at 6000 rpm for 10 min, washed three times successively with deionized water and anhydrous ethanol, and finally dried under vacuum to obtain copper-tannic acid nanoparticles (CuTA NPs).

[0037] c) Preparation of spermidine solutions of different concentrations Transfer 30 µL of spermidine (SPD) stock solution to 161 µL of pH = 7.4 PBS buffer and mix well to obtain a 1.0 M SPD solution.

[0038] Transfer 100 µL of 1.0 M SPD solution to 100 µL of pH 7.4 PBS buffer and mix well to obtain a 500 mM SPD solution.

[0039] Transfer 50 µL of 1.0 M SPD solution to 900 µL of pH 7.4 PBS buffer and mix well to obtain a 100 mM SPD solution.

[0040] d) Preparation of needle tip solution 10 mg of LAP powder was weighed and dissolved in 10 mL of pH 7.4 PBS buffer. The solution was stirred at 50°C in the dark for 15 min to ensure complete dissolution, yielding a 0.1% (w / v) LAP solution. Subsequently, 0.8 g of HAMA was weighed and added to the LAP solution, and stirring was continued in the dark to obtain an 8% (w / v) HAMA solution. Finally, 100 μL of the above-mentioned spermidine solutions of different concentrations (100 mM, 500 mM, and 1 M) were added respectively, and the solutions were stirred overnight at 37°C to obtain SPD / HAMA tip solutions containing different concentrations of spermidine (final SPD concentrations in the tip solutions were 1 mM, 5 mM, and 10 mM, respectively).

[0041] e) Preparation of base liquid Under room temperature and light-protected conditions, 50 mg of CuTA NPs were added to 10 mL of pH 7.4 PBS buffer. After multiple sonication treatments, a uniformly dispersed CuTA dispersion with a concentration of 5.0 mg / mL was obtained. Separately, 0.7 g of hyaluronic acid was added to 9.4 mL of pH 7.4 PBS buffer and stirred until completely dissolved. Then, 0.6 mL of the above CuTA dispersion was added, and the mixture was stirred overnight until homogeneous, yielding the CuTA / HA base solution. The final concentration of HA in the resulting base solution was 7% (w / v), and the final concentration of CuTA NPs was 0.3 mg / mL.

[0042] f) Fabrication of multifunctional microneedles with core-shell structure The tip solution SPD / HAMA prepared in step d was injected into the microneedle mold and centrifuged at 6000 rpm for 10 min to eliminate air bubbles and ensure the tip solution fully fills the micropores of the mold. After removing excess liquid from the mold surface, the mold was placed in a 30°C drying oven for 1 h. The above concentration steps were repeated three times to load more active ingredients onto the microneedle tips. Subsequently, the tip solution was irradiated with 365 nm ultraviolet light to initiate a free radical polymerization reaction of methacryloyl hyaluronic acid, forming a covalent cross-linked network. 0.5 g of the base solution CuTA / HA prepared in step e was injected into the mold. Finally, the mold was dried at 30°C for 24 h. After demolding, a multifunctional microneedle with a core-shell structure was obtained, denoted as CuTA / HA-SPD / HAMA (abbreviated as MN@CuTA+SPD). Example

[0043] In this embodiment, to verify the core-shell structure of MN@CuTA+SPD, the tip solution and substrate solution were labeled with two dyes, fluorescein isothiocyanate (FITC) and rhodamine B (RB), respectively. The spatial distribution of the microneedles was observed using an inverted fluorescence microscope and a confocal fluorescence microscope. The specific steps are as follows: a) Preparation of FITC-labeled spermidine solution Weigh 29.05 mg of fluorescein isothiocyanate (FITC) and dissolve it in 0.5 mL of dimethyl sulfoxide. After complete dissolution, add 2 mL of a 50 mM spermidine (SPD) solution prepared in Example 2 (SPD to FITC mass ratio approximately 1:2). Place the reaction system under light-protected conditions and stir for 24 h to obtain a fluorescein isothiocyanate-labeled spermidine (FITC-SPD) solution, which is then stored at 4°C in the dark for later use.

[0044] b) Preparation of FITC-labeled tip solution Weigh 10 mg of photoinitiator LAP powder and dissolve it in 10 mL of PBS buffer (pH = 7.4). Stir at 50°C in the dark for 15 min until completely dissolved to obtain a 0.1% (w / v) LAP solution. Then, add 0.8 g of HAMA and 10 μL of the FITC-SPD solution prepared in step a to the above solution and stir until homogeneous to obtain the FITC-labeled tip solution (FITC-SPD / HAMA).

[0045] c) Preparation of RB-labeled substrate solution 5.0 mg of Rhodamine B (RB) was dissolved in 10 mL of PBS buffer (pH = 7.4) to prepare a 0.5 mg / mL RB aqueous solution. Simultaneously, under light-protected conditions at room temperature, 50 mg of CuTA NPs were added to 10 mL of pH = 7.4 PBS buffer, and the solution was subjected to multiple sonication treatments to obtain a uniformly dispersed CuTA dispersion with a concentration of 5.0 mg / mL. Separately, 0.7 g of hyaluronic acid was added to 9.4 mL of pH = 7.4 PBS buffer and stirred until completely dissolved. Then, 0.1 mL of a 0.5 mg / mL RB aqueous solution and 0.6 mL of a 5.0 mg / mL CuTA dispersion were added, and the mixture was stirred overnight to obtain the RB-labeled base solution (RB-CuTA / HA).

[0046] d) Preparation of dual-dye labeled microneedles: The operation method is the same as step f in Example 2, except that the tip solution SPD / HAMA is replaced with the tip solution FITC-SPD / HAMA, and the base solution CuTA / HA is replaced with the base solution RB-CuTA / HA.

[0047] The prepared dual-dye labeled microneedles were dispersed in a buffer solution at pH 7.4, and their structure was observed using an inverted fluorescence microscope. The results are as follows: Figure 2 As shown in the image, bright-field images reveal that the microneedles are conical in shape with sharp tips and an intact overall structure. Simultaneously, the grayscale differences within the needle body region visually reveal its core-shell layered structure. Furthermore, fluorescence images show uniform, bright green fluorescence within the needle body under the FITC channel, while non-uniform red fluorescence is observed under the RB channel. This may be attributed to multiple interactions between Rhodamine B and CuTA NPs, including electrostatic and metal-coordination interactions, resulting in some Rhodamine B being loaded onto the CuTA NPs.

[0048] The above results demonstrate that the present invention has successfully prepared core-shell structured microneedles with potential sequential drug delivery capabilities.

[0049] Application Example 1 This application example aims to evaluate the cytotoxicity of the multifunctional microneedles (MN@CuTA+SPD) with a core-shell structure prepared in Example 2 at the in vitro cellular level.

[0050] According to ISO 10993-12 standard, sterile microneedle samples MN (blank microneedles without CuTA NPs and SPD loading), MN@CuTA (microneedles loaded with CuTA NPs), and MN@CuTA+SPD were respectively immersed in complete culture medium (90% DMEM / F12 basal medium, 10% fetal bovine serum, and 1% streptomycin-penicillin) and extracted at 37℃ for 72 h. After sterilization, the extracts were filtered through a 0.22 μm sterile filter membrane to obtain extracts of different microneedle materials. Human umbilical vein endothelial cells (HUVECs) were collected at 1.0 × 10⁻⁶... 5 Cells were seeded at a density of 1.0 mL / well in 12-well plates and incubated overnight at 37°C with 5% CO2 to allow cell adhesion. Subsequently, 1.0 mL of the corresponding extract was added to each of the experimental groups (MN group, MN@CuTA group, and MN@CuTA+SPD group), while the blank control group received an equal volume of fresh complete culture medium. After co-incubation of cells and material extracts for 24 h, cell viability was assessed using the CCK-8 assay.

[0051] Experimental results are as follows Figure 3 As shown, the cell viability of the blank microneedles (MN group) without CuTA NPs and SPD loading reached 111.3±0.9%, indicating that the biopolymer material used to construct the microneedles not only had no significant toxicity to HUVECs but also promoted cell proliferation to some extent. For the MN@CuTA+SPD group, as the spermidine concentration increased from 0 mM to 10 mM, the cell viability decreased from 108.4±0.7% to 85.5±1.2%, showing a dose-dependent trend. However, even at the highest concentration, the cell viability remained above 80%.

[0052] The above results indicate that the multifunctional microneedles MN@CuTA+SPD with a core-shell structure prepared in Example 2 have no obvious toxicity to HUVECs and exhibit good biocompatibility.

[0053] Application Example 2 This application example aims to evaluate the bactericidal effect of MN@CuTA+SPD prepared in Example 2 on Escherichia coli.

[0054] The frozen bacterial strain was inoculated into LB liquid medium and cultured at 37°C and 200 rpm on a shaker until the logarithmic growth phase (OD200). 600≈ 0.75). Working bacterial solutions were obtained by dilution with sterile physiological saline. Sterile microneedle samples (MN, MN@CuTA, and MN@CuTA+SPD) were placed in 24-well plates, with wells without microneedle samples serving as the blank control group (Blank group). 100 μL of working bacterial solution was added to each well, and the plates were incubated at 37°C for 8 h. After incubation, an equal volume of sterile physiological saline was added to each well, and the mixture was thoroughly mixed and the bacterial solution was collected. The bacterial solution was diluted to an appropriate multiple and spread onto LB agar plates. After incubation at 37°C for 18-24 h, bacterial counts were performed, and the bacterial viability of each group was calculated.

[0055] Experimental results are as follows Figure 4 As shown, the survival rate of *E. coli* in the MN group exceeded 100%, which is likely because the hyaluronic acid released from the degradation of microneedles provides nutrients for bacterial growth. In contrast, the survival rate of *E. coli* in the MN@CuTA group decreased to 10.1±0.2%, mainly attributed to the synergistic effect of Cu²⁺ and tannic acid released after microneedle degradation, which effectively disrupts the bacterial cell membrane, thereby killing most bacteria. Further introduction of SPD into the MN@CuTA microneedles reduced the survival rate of *E. coli* to 0.9±0.1%, indicating that the introduction of SPD not only did not weaken the antibacterial effect of the microneedles, but may have significantly improved antibacterial efficacy through multiple mechanisms, such as enhancing the electrostatic interaction between the microneedles and bacteria and promoting bacterial uptake of Cu²⁺.

[0056] The above results confirm that the MN@CuTA+SPD microneedles with a core-shell structure have good antibacterial properties and have the potential to be applied to the treatment of chronic diabetic wounds.

[0057] Application Example 3 This application example aims to establish a full-thickness skin defect model in Sprague-Dawley (SD) rats with type 2 diabetes and to evaluate the promoting effect of MN@CuTA+SPD on the skin wound healing process in Example 2. The specific steps are as follows: a) Establishment of a type 2 diabetic SD rat model Six-week-old male SPF-grade SD rats (weighing 200-220 g) were used. After fasting for 24 hours with unlimited access to water, streptozotocin (65 mL / kg, prepared with citrate buffer) was injected intraperitoneally to induce diabetes. Tail vein blood glucose was monitored for one week. If the blood glucose level remained above 16.7 mM, the model was considered successfully established.

[0058] b) Construction of a diabetic full-cortical defect model The successfully modeled diabetic rats were anesthetized (intraperitoneal injection of tribromoethanol, 10 mg / kg), and the hair on their backs was removed with depilatory cream. After disinfection with povidone-iodine, four circular full-thickness skin defects with a diameter of approximately 10 mm were cut on the back with surgical scissors, and the distance between the wounds was greater than 2 cm.

[0059] c) Wound treatment Sterile microneedle samples were placed on the wound surface, including the MN, MN@CuTA and MN@CuTA+SPD groups, with the untreated wound surface serving as the blank control group.

[0060] d) Postoperative observation and sampling The skin wound was photographed and recorded on postoperative days 0, 3, 7, 11, 15 and 19; the rats were sacrificed on day 19, and the wound and surrounding tissues were collected for histological staining analysis.

[0061] To evaluate the effects of different microneedle treatments on the healing of chronic diabetic wounds, this study performed hematoxylin-eosin (H&E) staining analysis on the wound and surrounding tissues. Figure 5 The results showed that, compared with the Blank group, the number of neovascularization (red arrows) in the wound tissue treated with MN@CuTA and MN@CuTA+SPD was significantly increased, and a large number of red blood cells were visible filling the lumen. This result indicates that MN@CuTA and MN@CuTA+SPD can promote angiogenesis in diabetic chronic wounds, and the abundant blood perfusion can provide sufficient oxygen and nutrients for subsequent tissue reconstruction, thus facilitating high-quality functional repair.

[0062] In summary, this invention proposes a multifunctional microneedle with a core-shell structure that integrates multiple functions such as anti-inflammatory, antibacterial, and angiogenesis promotion. This multifunctional microneedle can precisely regulate the microenvironment of diabetic chronic wounds through synergistic effects, providing a new solution for achieving high-quality wound healing.

[0063] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0064] Those skilled in the art will understand that the purpose of this invention is to provide a relatively universal overall preparation process or technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scale, product demand, etc.). Therefore, some specific operations can be flexibly adjusted according to the situation in actual implementation, as long as the expected or the same or similar technical effects as those in the embodiments of this invention can be achieved.

[0065] The steps in the method of this invention can be adjusted, combined, or deleted according to actual needs. The technical features can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the embodiments are described. However, as long as the combinations of these technical features do not contradict each other, they should all be considered within the scope of this invention.

[0066] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A multifunctional microneedle with a core-shell structure, characterized in that, Using methacrylic anhydride-modified biopolymer and spermidine as needle tip materials, the materials were centrifuged and filled into the micropores of a microneedle mold. The core layer of the microneedle was constructed by photo-initiated free radical polymerization of the methacrylic anhydride-modified biopolymer. Then, a mixture of polymer base material and metal-polyphenol nanomaterial was injected into the microneedle mold, dried and demolded to obtain a multifunctional microneedle with a core-shell structure.

2. The multifunctional microneedle with a core-shell structure according to claim 1, characterized in that, The methacrylic anhydride-modified biopolymer material is any one of methacrylamide hyaluronic acid, methacrylamide gelatin, methacrylamide collagen, methacrylamide carboxymethyl chitosan, and methacrylamide sodium alginate.

3. The multifunctional microneedle with a core-shell structure according to claim 1, characterized in that, The metal-polyphenol nanomaterial is any one of copper-tannic acid nanoparticles, iron-tannic acid nanoparticles, silver-curcumin nanoparticles, copper-epigallocatechin gallate nanoparticles, and iron-gallic acid nanoparticles.

4. The multifunctional microneedle with a core-shell structure according to claim 1, characterized in that, The polymer substrate material is any one of hyaluronic acid, polyvinyl alcohol, polyvinylpyrrolidone, polylactic acid, and polylactic acid-polyglycolic acid copolymer.

5. The multifunctional microneedle with a core-shell structure according to claim 1, characterized in that, The photoinitiator used for photoinitiation is either lithium phenyl (2,4,6-trimethylbenzoyl)phosphate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylphenylacetone.

6. A method for preparing a multifunctional microneedle with a core-shell structure, characterized in that, include: S1, Preparation of methacrylic anhydride-modified biopolymer materials: Biopolymer materials are dissolved in deionized water, and methacrylic anhydride is added dropwise to cause the methacrylic anhydride to undergo an acylation reaction with the amino or hydroxyl groups in the biopolymer materials. At the same time, sodium hydroxide solution is used to adjust the pH of the reaction solution. After the reaction is complete, impurities are removed by dialysis, and the biopolymer materials modified with methacrylic anhydride are obtained by freeze drying. S2, Preparation of metal-polyphenol nanomaterials: Metal ions and polyphenols were dissolved in deionized water, sodium hydroxide solution was added to adjust the pH of the reaction system, the mixture was stirred in a water bath, the precipitate was collected by centrifugation and washed with deionized water and ethanol, and the product was vacuum dried to obtain metal-polyphenol nanomaterials. S3, Preparation of needle tip solution: The photoinitiator was dissolved in PBS buffer and stirred in the dark to promote complete dissolution. Then, methacrylic anhydride-modified biopolymer and spermidine solution were added to obtain the needle tip solution. S4, Preparation of the base solution: Metal-polyphenol nanomaterials were dispersed in PBS buffer, followed by the addition of polymeric substrate material. The mixture was stirred until it was completely dissolved to obtain the substrate solution. S5, Preparation of multifunctional microneedles with core-shell structure: Inject the needle tip liquid into the microneedle mold, centrifuge to remove air bubbles and fill the micropores of the mold, and place the mold in a forced-air drying oven for concentration; The concentration steps were repeated multiple times. Then, the tip liquid was irradiated with ultraviolet light to initiate free radical polymerization of the methacrylic anhydride-modified biopolymer material to form a cross-linked network. Finally, the base liquid was added to the mold, and after drying and demolding, a multifunctional microneedle with a core-shell structure was obtained.

7. The method for preparing multifunctional microneedles with a core-shell structure according to claim 6, characterized in that, The reaction temperature in S1 is 4℃ ~ 50℃, and the reaction time is 2 h ~ 24 h; the reaction process in S2 is carried out at 50℃ under light-protected conditions; the ultraviolet irradiation time in S5 is 5 s ~ 60 s.

8. The method for preparing multifunctional microneedles with a core-shell structure according to claim 6, characterized in that, In the needle tip solution obtained in S3, the concentration of photoinitiator is 0.05 wt% ~ 0.3 wt%, the concentration of methacrylic anhydride modified biopolymer is 5 wt% ~ 15 wt%, and the concentration of spermidine is 1.0 mM ~ 10 mM.

9. The method for preparing multifunctional microneedles with a core-shell structure according to claim 6, characterized in that, In the substrate solution obtained in S4, the concentration of metal-polyphenol nanomaterials was 0.1 mg / mL ~ 0.5 mg / mL, and the concentration of polymer substrate material was 5 wt% ~ 40 wt%.

10. The application of a multifunctional microneedle with a core-shell structure as described in any one of claims 1 to 5, or a multifunctional microneedle with a core-shell structure prepared by any one of the preparation methods as described in claims 6 to 9, in the preparation of a material for healing chronic wounds in diabetic patients.

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