A soluble microneedle and a preparation method and application thereof

Abstract

This invention provides a soluble microneedle, its preparation method, and its application. The microneedle comprises a tip and a base. The tip is made of a composite material cross-linked with silk fibroin and tannic acid, and the base is made of a biocompatible material loaded with single-atom iron nanozymes. During preparation, the tip layer and base layer materials are separately filled into a microneedle mold, cured, dried, and then demolded. This microneedle employs a layered design; the tip layer can persistently release tannic acid for anti-inflammatory and antioxidant effects, while the base layer rapidly releases single-atom iron nanozymes in the inflammatory microenvironment, catalyzing the generation of reactive oxygen species for highly efficient antibacterial activity. The microneedle of this invention can simultaneously act on infection, inflammation, and oxidative stress, significantly improving the treatment effect of bacterial keratitis, and has advantages such as good biocompatibility, high drug utilization, and safe and convenient use.

Description

Technical Field

[0001] This invention relates to drug delivery microneedles, and more particularly to a soluble microneedle, its preparation method, and its application. Background Technology

[0002] Infectious keratitis is one of the leading causes of vision impairment and blindness worldwide. Its pathogenesis is closely related to the invasion of corneal tissue by pathogenic microorganisms such as bacteria and fungi. Epidemiological studies show that this disease causes more than one million cases of corneal blindness globally each year, and its incidence continues to rise. During the infection process, the level of reactive oxygen species (ROS) in the lesion area is significantly elevated, reaching 10-15 times that of normal corneal tissue. Excessive ROS significantly amplifies the inflammatory cascade by activating the NF-κB signaling pathway, while inhibiting the phagocytic function of macrophages and the regenerative capacity of corneal stem cells. This creates a mutually reinforcing and continuously worsening pathological cycle of "pathogen infection—inflammatory response—oxidative stress," exacerbating corneal tissue damage and delaying the repair process.

[0003] Currently, the clinical treatment of infectious keratitis mainly relies on two methods: topical eye drops and intravitreal injection. However, both have significant limitations. Topical administration is affected by multiple physiological barriers, including the tight junctions of the corneal epithelium, tear dilution, and nasolacrimal duct drainage, resulting in an actual bioavailability of less than 5% within the corneal tissue. To maintain the required effective concentration, frequent administration is necessary, which not only significantly increases the patient's medication burden and reduces treatment adherence but may also induce drug resistance in pathogens. In contrast, while intravitreal injection can increase local drug concentration in the short term, it is an invasive procedure with risks such as corneal perforation, hemorrhage, and secondary infection. Furthermore, it requires highly skilled operators and medical personnel, limiting its widespread clinical application.

[0004] Microneedling, as a novel local drug delivery method, has garnered widespread attention in the biomedical field in recent years due to its advantages such as being minimally invasive, painless, and easy to operate. Microneedles typically consist of an array of micron-sized needles that establish stable drug delivery channels while avoiding damage to deep nerves and blood vessels, thereby significantly improving drug bioavailability in target tissues and enhancing patient compliance. Furthermore, by optimizing the design of microneedle length and mechanical properties, and considering the characteristics of corneal tissue, repairable microchannels can be formed in the corneal epithelium, enabling precise delivery and sustained release of drugs into the corneal stroma.

[0005] Tannic acid (TA) is a widely sourced natural plant polyphenol compound. Its molecular structure is rich in catechol groups, which endow it with excellent biocompatibility, antioxidant, and anti-inflammatory activities. The abundant pyrogallol and catechol structures in TA can form strong non-covalent or coordination interactions with various matrix materials, and its hydrophobic aromatic ring structure also helps to repel interfacial water molecules, thereby enhancing the structural stability of the material system. However, TA's high water solubility in physiological environments leads to rapid elution, limiting its application in long-term local treatment and making it difficult to stably load into microneedle systems.

[0006] In the field of antibacterial therapy, nanozymes are considered a potential new antibacterial strategy to replace traditional antibiotics due to their advantages such as broad-spectrum antibacterial activity, high biocompatibility, and low likelihood of inducing drug resistance. Traditional nanozymes typically rely on catalyzing hydrogen peroxide to generate reactive oxygen species to kill pathogens, but their catalytic efficiency is easily affected by nanoparticle aggregation, resulting in insufficient exposure of active sites and a utilization rate of less than 30%.

[0007] For the reasons mentioned above, it is necessary to develop a new microneedle technology to solve at least one of the aforementioned technical problems. Summary of the Invention

[0008] This invention provides a soluble microneedle and its preparation method, as well as its application in the treatment of bacterial keratitis, to solve at least one of the above-mentioned technical problems.

[0009] According to a first aspect of the present invention, a soluble microneedle is provided, comprising a tip and a substrate, characterized in that the tip is prepared from a composite material formed by crosslinking silk fibroin (SF) and tannic acid (TA), and the substrate comprises a biocompatible material.

[0010] In a preferred embodiment, the substrate is prepared using a biocompatible material as a matrix to support single-atom iron nanozymes.

[0011] In a preferred embodiment, the composite material contains 20 wt% silk fibroin, 10 wt% tannic acid, and the remainder is deionized water.

[0012] In a preferred embodiment, the biocompatible material in the material has a mass fraction of 10 wt%, and the single-atom iron nanozyme has a loading concentration of 0.2 mg / mL in the hyaluronic acid solution.

[0013] In a preferred embodiment, the biocompatible material is selected from at least one of hyaluronic acid, methacrylamide gelatin, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol.

[0014] According to a second aspect of the present invention, the present invention also provides a method for preparing soluble microneedles, the method comprising the following steps: S1. A composite material formed by cross-linking silk fibroin (SF) and tannic acid (TA) is filled into the tip of a microneedle mold, cured in situ, and then dried to obtain the tip. S2. A material consisting of hyaluronic acid (HA) as a matrix to support single-atom iron nanozymes is filled into the base portion of a microneedle mold, and the base is obtained after drying. S3. After demolding, the soluble microneedles are obtained.

[0015] In a preferred embodiment, step S1 includes the following steps: S11. To prepare degummed silk fibroin from silkworm cocoons, the degummed silk fibroin is dissolved in a lithium bromide solution and heated to obtain a silk fibroin protein solution; S12. Add glycidyl methacrylate solution to silk fibroin solution and stir to react to achieve methacrylate esterification of silk fibroin. After the reaction is completed, filter to obtain filtrate, and dialyze the filtrate using a dialysis bag with a molecular weight cutoff of 12–14 kDa to obtain dialysate. Freeze the dialysate and then freeze-dry it to obtain methacrylated silk fibroin powder. Dissolve the methacrylated silk fibroin powder in deionized water, add photoinitiator LAP (lithium phenyl (2,4,6-trimethylbenzoyl)phosphate, CAS No. 85073-19-4) and tannic acid, and stir until completely dissolved to obtain TA-SF crosslinking precursor solution. S13. Inject the TA-SF crosslinking precursor solution into the tip of the microneedle mold. Use a vacuum-assisted method to fill the tip with the TA-SF crosslinking precursor solution. After removing air bubbles, irradiate to solidify the system in situ. Dry overnight to obtain the tip of the microneedle.

[0016] In a preferred embodiment, step S2 includes the following steps: S21. Synthesis of single-atom iron nanozymes: 2-methylimidazole and heme are dissolved in methanol to form solution A; Solution A was dissolved in methanol to form solution B. Solution A and solution B were mixed and stirred at room temperature. The product was collected by centrifugation, washed with methanol and dried under vacuum overnight to obtain the Fe / ZIF-8 precursor. Fe / ZIF-8 and 5-benzimidazolyl-1,3-phthalic acid were dispersed in methanol, reacted at room temperature, centrifuged, washed and dried. The product was heat-treated at a predetermined heating rate for a predetermined time under a high-temperature nitrogen atmosphere and then naturally cooled to obtain the single-atom iron nanozyme Fe-NC. S22. Weigh out hyaluronic acid and dissolve it in deionized water to obtain a 10% hyaluronic acid solution; then add the single-atom iron nanozyme Fe-NC obtained in step S1 to the solution, stir thoroughly, and then sonicate it to disperse it fully, finally obtaining a hyaluronic acid / single-atom iron nanozyme mixed solution HA-SACs; S23. The hyaluronic acid / single-atom iron nanoenzyme mixed solution HA-SACs obtained in step S2 is poured into the base part of the upper microneedle mold, and then dried after vacuum degassing to obtain the base of the microneedle.

[0017] According to a third aspect of the present invention, the present invention also provides a soluble microneedle patch comprising an array of a plurality of the aforementioned soluble microneedles.

[0018] According to a fourth aspect of the present invention, the present invention also provides a soluble microneedle for treating bacterial keratitis, characterized in that the microneedle is the aforementioned soluble microneedle.

[0019] In a preferred embodiment, the tip height is 400 micrometers and the substrate height is 250 micrometers. Attached Figure Description

[0020] Figure 1 Example 1: Schematic diagram of the soluble microneedle structure; Figure 2 Characterization diagram of the single-atom iron nanozyme Fe-NC in Example 3; Figure 3 Verification diagram of the single-atom iron nanozyme in Example 4 against Gram-negative bacteria Escherichia coli and Gram-positive bacteria Staphylococcus aureus in vitro; Figure 4 Cell membrane characterization diagram after treatment with single-atom iron nanozymes in Example 4; Figure 5 The in vitro antioxidant performance verification diagram of tannic acid in Example 5; Figure 6 Characterization diagram of the effect of soluble microneedles loaded with tannic acid on cell migration ability; Figure 7 Characterization diagram of soluble microneedle patch; Figure 8 Characterization diagram of the sustained-release performance of the MN patch on the ocular surface in Example 7; Figure 9 Characterization diagram of the animal experiment of soluble microneedles for treating keratitis in Example 8; Figure 10 Comparison of mechanical-displacement tests on tannic acid loaded on different materials. Detailed Implementation

[0021] The information regarding the experimental animals and reagents involved in this application is as follows: Experimental cell lines: Human corneal epithelial cells (HCECs), purchased from the American Type Culture Collection (ATCC); mouse mononuclear macrophage cell line RAW 264.7, purchased from the National Type Culture Collection (NTCC).

[0022] Experimental animals: Wistar female rats (4-6 weeks old, weighing 200-250 g) were purchased from Spiford (Beijing) Biotechnology Co., Ltd.

[0023] Main experimental materials: Tannic acid (TA): Product No.: 1401-55-4, Shanghai Aladdin Biochemical Technology Co., Ltd.; Hyaluronic acid (HA): Product No.: 9067-32-7, Shanghai Maclean Biochemical Technology Co., Ltd.

[0024] Example 1: Soluble microneedles refer to Figure 1 This application provides a soluble microneedle 1, comprising a needle tip 11 and a substrate 12, wherein the needle tip is prepared from a composite material formed by crosslinking silk fibroin (SF) and tannic acid (TA). In one specific embodiment, the composite material, calculated on a total mass fraction basis of 100%, comprises 20 wt% silk fibroin, 10 wt% tannic acid, and the balance being deionized water. In this embodiment, the substrate may be composed of components such as biocompatible materials to support the needle tip. The biocompatible material may be selected from at least one of hyaluronic acid, methacrylamide gelatin, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol.

[0025] In this approach, a silk fibroin-tannic acid composite is used to prepare the microneedle tip, balancing the mechanical properties of the microneedle with sustained anti-inflammatory and antioxidant functions. Specifically, the amide bonds (-CONH-) in the silk fibroin can form a stable hydrogen bond network with the phenolic hydroxyl groups (-OH) in the tannic acid molecule, thereby significantly enhancing the mechanical strength of the microneedle tip. Simultaneously, the silk fibroin undergoes self-assembly during cross-linking, forming a high-density cross-linked network rich in β-sheet structures. This effectively prevents the rapid elution of tannic acid in the physiological environment, achieving sustained release of anti-inflammatory and antioxidant components, significantly improving the local corneal inflammation and oxidative stress microenvironment, enhancing the persistence of anti-inflammatory and antioxidant effects, promoting corneal repair, and improving prognosis. Furthermore, the self-assembly of silk fibroin... The folded structure enhances the mechanical strength of the microneedles, ensuring their smooth penetration of the corneal epithelium.

[0026] In a preferred embodiment, substrate 12 is prepared using hyaluronic acid (HA) as the matrix for loading single-atom iron nanozymes. In one specific embodiment, the hyaluronic acid content is 10 wt%, the loading concentration of single-atom iron nanozymes in the biocompatible material is 0.2 mg / mL, and the remainder is deionized water, calculated on a total mass fraction of 100%.

[0027] In this preferred embodiment, the combination of single-atom iron nanozymes (SACs) and microneedle delivery technology significantly improves the local antibacterial efficiency of the cornea and reduces the risk of drug resistance. Specifically, hyaluronic acid (HA) is a linear polysaccharide naturally found in ocular surface tissues, exhibiting excellent hydrophilicity and environmental responsiveness. Inflammatory lesions typically present a weakly acidic environment, which can induce accelerated degradation of the hyaluronic acid matrix, thereby rapidly releasing the loaded single-atom iron nanozymes. The released SACs catalyze the generation of high concentrations of reactive oxygen species (ROS), which oxidatively destroy the lipopolysaccharides and protein structures of bacterial cell walls, achieving a broad-spectrum antibacterial effect with an inhibition rate exceeding 99%. Compared to treatments relying on traditional antibiotics, this application achieves a broad-spectrum, highly effective, and less resistant-to-drug-resistant antibacterial effect through the efficient catalysis of ROS by single-atom iron nanozymes; simultaneously, it overcomes the limitation of existing technologies where drugs have difficulty effectively penetrating corneal tissue by delivering the drugs directly to the corneal lesion area via microneedles.

[0028] As previously described, this application discloses a soluble microneedle comprising a needle tip 11 and a substrate 12. The needle tip is prepared from a composite material formed by cross-linking silk fibroin (SF) and tannic acid (TA), while the substrate can be prepared using hyaluronic acid (HA) as a matrix to support single-atom iron nanozymes. In this approach, the soluble microneedle possesses a well-defined sequential therapeutic mechanism, enabling synergistic intervention against the core pathological features of infectious keratitis. Specifically, addressing the problem of fragmented antibacterial and anti-inflammatory treatments in existing technologies, which hinder simultaneous efficacy, this application innovatively utilizes a spatially layered microneedle structure to achieve a programmed treatment strategy of "rapid antibacterial treatment followed by sustained anti-inflammatory and antioxidant effects." This effectively blocks the vicious cycle of infection-inflammation-oxidative stress, fundamentally improving the treatment efficacy of infectious keratitis. Meanwhile, the materials used are all biocompatible natural polymers or functional nanomaterials. The microneedle preparation process is mature and easy to operate. The mechanical properties and release behavior can be flexibly adjusted by controlling the material ratio and structural parameters. The minimal corneal trauma caused by the microneedles can heal spontaneously in a short time, making it suitable for repeated drug administration. This provides a safe, effective, and promising new treatment option for infectious keratitis. Example 2: Preparation method of soluble microneedles 1. Preparation of the silk fibroin / tannic acid cross-linking system TA-SF (TS) 40 g of chopped silkworm cocoons were placed in 1 L of 0.05 M Na₂CO₃ solution and boiled at 100 °C for 30 min to remove sericin. The mixture was then thoroughly washed with distilled water and dried at room temperature. 20 g of degummed silk fibroin was dissolved in 100 mL of 9.3 M lithium bromide (LiBr) solution and heated at 60 °C for 1 h to obtain a silk fibroin solution. 6 mL of glycidyl methacrylate (GMA) solution was added to this solution, and the mixture was stirred at 60 °C and 300 rpm for 3 h to achieve methacrylate esterification of the silk fibroin. After the reaction, the solution was filtered through a Miracloth filter and dialyzed against distilled water for 4 days using a dialysis bag with a molecular weight cutoff of 12–14 kDa. The resulting solution was then frozen at -80 °C for 12 h and lyophilized for 48 h to obtain methacrylated silk fibroin (SilMA) powder.

[0029] Dissolve 0.29 g SilMA in 1 mL of deionized water and stir at 300 rpm for 3 h at room temperature. Add 2.5 mg of photoinitiator LAP and 0.14 g of tannic acid and continue stirring until completely dissolved to obtain the TA-SF crosslinking precursor solution.

[0030] 2. Preparation of hyaluronic acid / single-atom iron nanozyme mixed solution HA-SACs Synthesis of single-atom iron nanozymes: 2.36 g of 2-methylimidazole and 60 mg of heme were dissolved in 40 mL of methanol to form solution A; Solution A was dissolved in 40 mL of methanol to form solution B. Solution A and solution B were mixed and stirred at room temperature for 24 h. The product was collected by centrifugation, washed with methanol, and dried under vacuum at 70 °C overnight to obtain the Fe / ZIF-8 precursor. Subsequently, Fe / ZIF-8 was dispersed with 60 mg of 5-bop (5-benzimidazolyl-1,3-phthalic acid) in methanol, reacted at room temperature, washed by centrifugation, and dried. The resulting product was then dried under a nitrogen atmosphere at 930 °C. Heat treatment at a heating rate of 2 h was performed, followed by natural cooling to obtain single-atom iron nanozyme Fe-NC (see Example 3 for specific characterization).

[0031] Weigh 5 g of hyaluronic acid and add it to 45 mL of deionized water. Stir at 300 rpm at room temperature until completely dissolved to obtain a 10% hyaluronic acid solution. Then add 10 mg of Fe-NC single-atom nanozyme to the solution, stir at 300 rpm for 1 h, and use sonication to fully disperse it, finally obtaining a hyaluronic acid / single-atom iron nanozyme mixed solution HA-SACs.

[0032] 3. Preparation of soluble microneedles The TA-SF crosslinking system was injected into a PDMS microneedle mold, and the microneedle tips were filled using a vacuum-assisted method. After removing air bubbles, the system was cured in situ by irradiation with 405 nm blue light and dried overnight at 45 °C to form microneedle tips. Subsequently, a HA-SACs mixed solution was poured onto the upper layer of the mold to construct the microneedle substrate. After vacuum degassing, it was dried again at 45 °C, and finally demolded to obtain a programmed bilayer soluble microneedle patch.

[0033] Example 3: Characterization of single-atom iron nanozymes (Fe-NC materials) Single-atom iron nanozymes (Fe-NC) were prepared according to the method in Example 2. (Reference) Figure 2 Scanning electron microscopy (SEM) results showed that the obtained Fe-NC retained the rhombic dodecahedral morphology of the ZIF-8 precursor. Energy dispersive spectroscopy (EDS) analysis indicated that C, N, and Fe elements were uniformly distributed in the material, and no obvious metal agglomeration was observed. X-ray photoelectron spectroscopy (XPS) analysis showed that in the N 1s energy level spectrum, Fe-NC corresponded to pyridine nitrogen, pyrrole nitrogen, graphitic nitrogen, and nitrogen oxide structures at approximately 398.2 eV, 399.8 eV, 401.0 eV, and 405.3 eV, respectively, indicating that iron atoms were distributed in the Fe-N configuration.x The coordination form is stably anchored in the carbon-based support.

[0034] The enzyme-like catalytic performance of the prepared Fe-NC was evaluated by UV-Vis absorption spectroscopy of three typical chromogenic substrate systems (TMB, OPD, and ABTS). The results showed that, in the presence of H₂O₂, Fe-NC could effectively catalyze the oxidation reactions of the three substrates, with peak values ​​at approximately 652 nm (ox-TMB), 450 nm (ox-OPD), and [other substrates not specified in the original text]. The presence of a significantly enhanced characteristic absorption peak indicates the rapid formation of the corresponding oxidation product; in the control system lacking H2O2 or Fe-NC, only a very weak absorption signal was observed. Furthermore, Fe-NC exhibited significantly higher catalytic activity than N–C in various substrate systems. These results demonstrate that Fe-NC possesses broad-spectrum and stable peroxidase-like (POD-like) catalytic activity.

[0035] Example 4: Validation of the in vitro antibacterial performance of single-atom iron nanozyme Fe-NC The antibacterial properties and mechanism of action of Fe-NC single-atom nanozymes were systematically evaluated using various in vitro experiments. Gram-negative bacteria *Escherichia coli* (E. coli) were used as the starting material. E. coli ) and Gram-positive bacteria Staphylococcus aureus ( S. aureus The model strain was selected. First, Fe-NC suspensions at different final concentrations (0, 50, 100, 150, 200, 250 µg / mL) were added to a solution containing 10... 6 In bacterial culture tubes containing CFU / mL, Under the specified conditions, the cells were cultured for 3 hours. After the culture was completed, the cells were harvested. Spread the bacterial culture evenly onto a nutrient agar plate and place it in a constant temperature incubator. After overnight incubation, colony counts were performed. The results showed that Fe-NC achieved significant antibacterial activity at a final concentration of 200 μg / mL, which was determined as the minimum inhibitory concentration. Subsequently, bacterial morphology was characterized using scanning electron microscopy (SEM), referencing... Figure 3 As can be seen, compared with the control group, the bacterial cell membrane structure was significantly damaged after Fe-NC treatment, accompanied by leakage of cell contents, indicating that the material can directly destroy the structural integrity of bacteria.

[0036] To investigate its antibacterial mechanism, DCFH-DA was used as a fluorescent probe, and flow cytometry was used to detect the generation of reactive oxygen species (ROS) in the bacterial system. (Reference) Figure 4The results showed that the ROS level in the Fe-NC + H2O2 treatment system was significantly higher than that in the control group and the NC + H2O2 group, indicating that Fe-NC has excellent peroxidase-like (POD-like) catalytic activity. Further analysis of bacterial biofilm structural changes was conducted using live / dead fluorescence staining combined with confocal laser microscopy (CLSM). The results showed that the biofilm continuity was significantly reduced and the proportion of dead cells significantly increased after Fe-NC treatment. Especially in the presence of H2O2, the biofilm structure was almost completely destroyed, exhibiting obvious red fluorescence, indicating that it can simultaneously achieve highly efficient bactericidal and anti-biofilm effects.

[0037] In summary, the in vitro experimental results show that Fe-NC single-atom nanozymes have efficient and broad-spectrum antibacterial and anti-biofilm properties.

[0038] Example 5: Validation of the in vitro antioxidant properties of tannic acid Using human corneal epithelial cells (HCECs) as a model, the regulatory effect of tannic acid (TA) on intracellular reactive oxygen species (ROS) levels was evaluated using a reactive oxygen species fluorescent probe (DCFH-DA). HCECs were tested at a concentration of 1.5 × 10⁻⁶. 5 Cells were evenly seeded at a density of [number] cells / mL in 12-well plates pre-coated with sterilized confocal microscopy slides and incubated overnight at 37 °C and 5% CO2. Subsequently, the medium was replaced with complete medium containing different treatment components and incubated for another 6 h. The original medium was discarded, and 10 μM DCFH-DA solution was added. The plates were incubated in the dark for 30 min. After fixation, nucleus staining, and mounting, intracellular fluorescence signals were observed using a laser confocal microscope. The results showed that, as reference [reference]... Figure 5 The green fluorescence intensity was positively correlated with the ROS level. Compared with the control group, the green fluorescence intensity of HCECs treated with TA was significantly reduced, indicating that TA can effectively inhibit the production of intracellular ROS and demonstrate good antioxidant potential.

[0039] Example 6: Validation of the antioxidant properties of tannic acid loaded on soluble microneedles To further evaluate the promoting effect of tannic acid (TA) on corneal epithelial repair after delivery via a microneedle system, a cell scratch migration assay was used to compare the migration ability of human corneal epithelial cells (HCECs) under different treatment conditions. A control group (blank culture medium), a hyaluronic acid microneedle extract group (HA group), a hyaluronic acid / silk fibroin microneedle extract group (HA-SF group), and a tannic acid-containing microneedle extract group (HA+TA-SF group) from the present invention were set up for comparison. The extracts used in each microneedle treatment group were prepared by incubating the corresponding microneedle patches in culture medium for 24 hours to simulate the actual drug release behavior in vivo.

[0040] HCECs were cultured in DMEM / F12 complete medium containing 10% fetal bovine serum and 1% penicillin and streptomycin. Cultured under constant temperature conditions. Cells in the logarithmic growth phase were cultured at approximately 1.5 × 10⁻⁶ cells / year. 5 Cells were seeded at a density of [number] cells / mL in 12-well plates. After reaching approximately 90% confluence, the original culture medium was discarded, and the cells were gently washed twice with sterile PBS to remove any floating cells. Subsequently, sterile [treatment / treatment] was performed. With the pipette tip perpendicular to the bottom of the well plate, a linear scratch was made evenly in the center of the cell monolayer. After scratching, the cells were washed twice with PBS to remove detached cells and cell debris. Then, the corresponding treatment medium was added to each well. Cell morphology in the scratched area was recorded using an inverted microscope at 0 h. The cells were then cultured in an incubator for another 24 h, and microscopic images were acquired again at the same location to record cell migration to the scratched area.

[0041] refer to Figure 6 The experimental results showed that after 24 h of culture, the cells in the PBS group had weak migration ability, and a large area of ​​the scratched region remained unclosed. Cell migration improved in the HA and HA+SF groups, suggesting that SF can promote corneal epithelial repair by providing a favorable microenvironment for cell adhesion and migration; however, gaps not covered by cells were still clearly visible in the scratched region. In contrast, the scratched region in the HA+TA-SF group was significantly reduced, and the migration of corneal epithelial cells towards the scratch center was significantly enhanced, with denser cell arrangement, demonstrating excellent migration and repair capabilities.

[0042] The above results indicate that tannic acid, delivered via a microneedle system, can not only effectively inhibit inflammatory responses and reactive oxygen species generation, but also significantly promote the migration and repair of corneal epithelial cells, which is beneficial for the rapid healing of damaged corneal tissue.

[0043] Example 7: Characterization of soluble microneedle patches Corneal microneedle (MN) patches are prepared using a casting method. The specific preparation method can be the existing casting method, which is well-known in the art and will not be elaborated upon here. (Reference) Figure 7 The resulting MN patch consisted of 68 microneedles arranged in a regular circular array, with each needle having a height of 650 μm. Scanning electron microscopy (SEM) results showed that the microneedles had consistent morphology, intact structure, and sharp tips, indicating good reproducibility and molding stability of the fabrication process. To further verify the layered structure of MN, the red fluorescent probe Rhodamine B and the green fluorescent probe FITC were introduced into the needle tip and substrate, respectively, during the fabrication process. Bright-field and fluorescence microscopy observations showed that the microneedles exhibited a layered structure with clear interlayer interfaces, laying the structural foundation for zoned delivery and functional design.

[0044] The MN patch exhibits high mechanical strength and successfully penetrates the rat corneal epithelium. Observation was performed using sodium fluorescein staining combined with slit-lamp cobalt blue light, as referenced. Figure 8 The micro-wounds caused by MN healed completely within 48 hours, with no obvious corneal opacity or persistent damage observed, validating the biosafety and feasibility of this microneedle patch for ocular drug delivery. Based on this, FITC-labeled bovine serum albumin (FITC-BSA) was used as a model drug, and the drug retention behavior on the ocular surface was evaluated using a small animal in vivo fluorescence imaging system. (Reference) Figure 8 The results showed that after administration via the MN patch, the fluorescence signal in the corneal region persisted for more than 12 hours, while the fluorescence signal in the conventional eye drop group rapidly decayed and disappeared within 10 minutes. These results indicate that the MN patch significantly improves the retention time of the drug on the corneal surface, thus potentially enhancing the bioavailability and therapeutic effect of ocular drug delivery.

[0045] To systematically evaluate the therapeutic effect of the bilayer microneedle patch of this invention in vivo, a rat model of bacterial keratitis was established and a comparative treatment study was conducted. Specifically, the microneedle patch prepared in Example 7 was inserted into the ocular surface for the experiment. Rats were randomly divided into four groups: control group, traditional antibiotic eye drop group, MN+Fe-NC group, and MN+Fe-N-C+TA-SF group. An infection model was established by injecting a mixed bacterial solution of Staphylococcus aureus and Escherichia coli into the corneal stroma. 24 hours after modeling, the rats showed obvious inflammatory symptoms such as corneal opacity, edema, and increased secretions, indicating successful model establishment. Subsequently, each group was treated continuously for 5 days according to the predetermined protocol, and corneal changes were recorded using a slit-lamp microscope on days 0, 1, 3, and 5 of treatment. After treatment, the animals were sacrificed, and corneal tissue was collected for H&E staining analysis to further evaluate corneal structural repair and inflammatory response at the histological level.

[0046] refer to Figure 9 The results showed that the model group had severe corneal inflammation, with significant disruption of the corneal hierarchical structure and extensive infiltration of inflammatory cells. While the inflammation in the eye drop group was somewhat relieved, the cornea remained significantly cloudy. In contrast, the MN+Fe-NC group showed a superior treatment trend, while the MN+Fe-N-C+TA group exhibited the most significant therapeutic effect, with a significant reduction in corneal cloudiness, near-complete restoration of corneal transparency, and significant suppression of the inflammatory response. Histological analysis showed that the corneal structure in this group had essentially returned to normal, with no significant edema or inflammatory cell infiltration observed. The combined slit-lamp observation and histological results indicate that the bilayer microneedle patch loaded with Fe-NC and TA demonstrated a significant synergistic effect of anti-infection and anti-inflammation in the treatment of bacterial keratitis, effectively promoting corneal tissue repair, and was significantly superior to traditional eye drop administration methods.

[0047] Example 9: Comparison of mechanical properties of tannic acid supported on different matrices To ensure comparability of mechanical properties, the formulations of PVA, PVP, and HA-supported tannic acid (TA) were all prepared using the method of "matrix material + TA + deionized water". The total mass fraction of the system was 100%, of which the matrix material was 20 wt%, TA was 10 wt%, and deionized water was 70 wt%.

[0048] Specifically, 2.0 g of polyvinyl alcohol (PVA) was added to 7.0 g of deionized water and stirred at 300 rpm for 3 h in a 90 ℃ water bath until the PVA was completely dissolved and the solution was clear and homogeneous. Then, the solution was cooled to room temperature, and 1.0 g of tannic acid (TA) was added. The solution was stirred at room temperature until it was completely dissolved to obtain a PVA-TA precursor mixed solution.

[0049] Add 2.0 g of polyvinylpyrrolidone (PVP) to 7.0 g of deionized water and stir at 300 rpm for 3 h at room temperature until PVP is completely dissolved and the solution is clear and homogeneous; add 1.0 g of tannic acid (TA) and continue stirring until completely dissolved to obtain a PVP-TA precursor mixed solution.

[0050] Take 2.0 g of hyaluronic acid (HA) and slowly add it to 7.0 g of deionized water. Stir at room temperature and fully hydrate until HA is completely dissolved to form a homogeneous system. Then add 1.0 g of tannic acid (TA) and stir at low speed until completely dissolved to obtain HA-TA precursor mixed solution.

[0051] The above-mentioned PVA-TA, PVP-TA and HA-TA precursor mixed solutions were injected into PDMS microneedle molds, and the microneedles were filled and debubbled simultaneously using a vacuum-assisted method. They were then dried overnight at 45 ℃. After drying, the molds were demolded to obtain PVA-TA, PVP-TA and HA-TA microneedle patches, respectively.

[0052] The mechanical properties of the microneedle patch were tested using a TA.XT Plus texture analyzer from Stable Micro Systems (SMS), UK. A planar indenter was used, with the microneedle patch fixed in the center of the sample stage, ensuring the indenter's downward pressure direction was aligned with the normal direction of the microneedle array. Compression tests were conducted at a compression rate of 2 mm / min, and the force-displacement data generated during compression were recorded in real time. The tested microneedle array had a single needle length of 650 μm and a total of 68 needles. The total force value obtained from the test was divided by the number of microneedles to convert it into a single needle force value (N / needle), and a force-displacement curve was plotted with the single needle force as the ordinate and displacement as the abscissa.

[0053] Under the above test conditions, force-displacement tests were performed on microneedle patches loaded with tannic acid in different matrix materials. The single-needle force-displacement curves are shown below. Figure 10 As shown in the figure. The results indicate that, under the same displacement conditions, there are significant differences in the single-needle load-bearing capacity of microneedles with different matrices. Among them, the silk fibroin-loaded tannic acid (SF-TA) microneedles exhibited the highest single-needle force value throughout the entire compression displacement range, with the largest slope of its force-displacement curve, indicating that it has the best mechanical stiffness and load-bearing capacity; the polyvinyl alcohol-loaded tannic acid (PVA+TA) microneedles were second, showing good mechanical support performance; the single-needle force of the polyvinylpyrrolidone-loaded tannic acid (PVP+TA) microneedles was further reduced; while the single-needle load-bearing capacity of the hyaluronic acid-loaded tannic acid (HA+TA) microneedles was the lowest. The single-needle force of each group of microneedles showed a non-linear increasing trend with the increase of displacement, and no obvious sudden drop in force value occurred within the test displacement range, indicating that the microneedle structure did not undergo sudden fracture during compression. The above results indicate that different matrix materials have a significant impact on the mechanical properties of microneedles, and silk fibroin as a matrix material is more conducive to improving the mechanical strength and structural stability of microneedles, thereby meeting the requirements of microneedles for insertion ability and deformation resistance in actual use.

[0054] Also refer to Figure 10 Force-displacement tests show that, under the same geometric dimensions, the load-bearing capacity of silk fibroin-based microneedles is significantly higher than that of microneedles made from common hydrophilic polymers such as polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), and hyaluronic acid (HA), especially exhibiting a faster load growth rate and higher initial stiffness within a small displacement range. This small displacement range corresponds to the critical stress stage when the microneedle penetrates the cornea, directly reflecting the penetration capability of the microneedle tip in practical applications. Based on the above results and the known mechanical strengthening effect of tannic acid on silk fibroin materials, this invention uses a silk fibroin-tannic acid composite system to construct the microneedle tip, which can further improve the mechanical strength and structural stability of the tip. This ensures stable penetration into corneal epithelial tissue while maintaining the overall biodegradability and biocompatibility of the microneedle, reducing the risk of microneedle bending or failure, and significantly improving the reliability and safety of ocular surface drug delivery.

[0055] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A soluble microneedle, comprising a needle tip and a base, characterized in that, The needle tip is made of a composite material formed by cross-linking silk fibroin and tannic acid, and the substrate includes a biocompatible material.

2. The soluble microneedles according to claim 1, characterized in that, The substrate is prepared using a biocompatible material as a matrix to support single-atom iron nanozymes.

3. The soluble microneedles according to claim 1, characterized in that, In the composite material, the mass fraction of silk fibroin is 20 wt%, the mass fraction of tannic acid is 10 wt%, and the balance is deionized water.

4. The soluble microneedles according to claim 2, characterized in that, The biocompatible material has a mass fraction of 10 wt%, and the loading concentration of single-atom iron nanozyme in the biocompatible material is 0.2 mg / mL.

5. The soluble microneedles according to any one of claims 2 or 4, wherein the biocompatible material is selected from at least one of hyaluronic acid, methacrylamide gelatin, polyvinyl alcohol, polyvinylpyrrolidone, and polyethylene glycol.

6. A method for preparing the soluble microneedles according to any one of claims 1-5, the method comprising the following steps: S1. The composite material formed by cross-linking silk fibroin and tannic acid is filled into the tip of the microneedle mold, and the tip is obtained by in-situ curing and drying. S2. The material containing hyaluronic acid as a matrix and supporting single-atom iron nanozymes is filled into the base portion of the microneedle mold and dried to obtain the base. S3. After demolding, the soluble microneedles are obtained.

7. The method for preparing soluble microneedles according to claim 6, characterized in that, Step S1 includes the following steps: S11. To prepare degummed silk fibroin from silkworm cocoons, the degummed silk fibroin is dissolved in a lithium bromide solution and heated to obtain a silk fibroin protein solution; S12. Add glycidyl methacrylate solution to silk fibroin solution and stir to react to achieve methacrylate esterification of silk fibroin. After the reaction is complete, filter to obtain filtrate, and dialyze the filtrate using a dialysis bag with a molecular weight cutoff of 12–14 kDa to obtain dialysate. Freeze the dialysate and then freeze-dry it to obtain methacrylated silk fibroin powder. Dissolve the methacrylated silk fibroin powder in deionized water, add photoinitiator LAP and tannic acid, and stir until completely dissolved to obtain TA-SF crosslinking precursor solution. S13. Inject the TA-SF crosslinking precursor solution into the tip of the microneedle mold. Use a vacuum-assisted method to fill the tip with the TA-SF crosslinking precursor solution. After removing air bubbles, irradiate to solidify the system in situ. Dry overnight to obtain the tip of the microneedle.

8. The method for preparing soluble microneedles according to claim 6, characterized in that, Step S2 includes the following steps: S21. Synthesis of single-atom iron nanozymes: 2-methylimidazolium and heme were dissolved in methanol to form solution A; Zn(NO3)2·6H2O was dissolved in methanol to form solution B. Solution A and solution B were mixed and stirred at room temperature. The product was collected by centrifugation, washed with methanol and dried under vacuum overnight to obtain the Fe / ZIF-8 precursor; Fe / ZIF-8 and 5-benzimidazolyl-1,3-phthalic acid were dispersed in methanol, reacted at room temperature, centrifuged, washed and dried. The obtained product was heat-treated at a predetermined heating rate for a predetermined time under a high-temperature nitrogen atmosphere and then naturally cooled to obtain the single-atom iron nanozyme Fe-NC. S22. Weigh out hyaluronic acid and dissolve it in deionized water to obtain a 10% hyaluronic acid solution; then add the single-atom iron nanozyme Fe-NC obtained in step S21 to the solution, stir thoroughly, and then sonicate it to disperse it fully, finally obtaining a hyaluronic acid / single-atom iron nanozyme mixed solution HA-SACs; S23. The hyaluronic acid / single-atom iron nanoenzyme mixed solution HA-SACs obtained in step S2 is poured into the base part of the upper microneedle mold, and then dried after vacuum degassing to obtain the base of the microneedle.

9. A soluble microneedle patch, characterized in that, The soluble microneedle patch comprises an array of multiple soluble microneedles, wherein the soluble microneedles are the soluble microneedles as described in any one of claims 1-5.

10. A soluble microneedle for treating bacterial keratitis, characterized in that, The microneedle is the soluble microneedle according to any one of claims 1-5.

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