Concave light-controlled microneedle for corneal anti-inflammatory and antibacterial and preparation method and application thereof

Abstract

The application provides a concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes, a preparation method and application, wherein the concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes comprises a concave transparent substrate and a soluble microneedle array formed on the concave surface of the substrate; each microneedle structure in the microneedle array comprises a needle body and a needle tip arranged at the top end of the needle body, and the needle tip is composed of a soluble polymer material and a light response compound; the light response compound comprises a hydroxyl-functionalized graphyne and silver nanoparticles loaded thereon, and can generate a photothermal and / or photodynamic effect under near-infrared light irradiation, so as to realize light-controlled release of drugs and synergistic anti-inflammatory and antibacterial effects. The application integrates high biocompatibility, light-controlled precise release, structure adaptation and synergistic anti-inflammatory and antibacterial effects, effectively solves the problems of poor conformability, drug burst release and single function of existing products, and has potential clinical application value in the treatment of eye diseases such as bacterial keratitis, postoperative corneal infection, corneal inflammation and corneal epithelial defect.

Description

Technical Field

[0001] This invention relates to the field of biomedical engineering, and in particular to a concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes, its preparation method, and its application. Background Technology

[0002] The cornea, with its intricate layered structure and immune function, is a crucial barrier for maintaining the integrity of the eyeball and visual acuity. Corneal diseases, especially infectious keratitis and postoperative inflammatory reactions, are common causes of blindness worldwide. Currently, routine treatment for these diseases mainly relies on frequent topical instillation of antibiotics and anti-inflammatory eye drops. However, this approach has significant limitations: firstly, the tight junctions of the corneal epithelium and the rapid flushing by tears result in extremely low ocular bioavailability of the drugs (usually less than 5%); secondly, frequent administration not only causes great inconvenience to patients but also easily leads to ocular surface damage and drug toxicity accumulation; more importantly, it is difficult to maintain a sustained and effective therapeutic concentration at the lesion site, which not only affects efficacy but may also lead to the development of bacterial resistance.

[0003] Emerging microneedle transdermal drug delivery technology offers a new approach to solving the aforementioned problems. Its core lies in utilizing a micron-scale array of needles to briefly penetrate the outermost barrier of the cornea (epithelial layer) in a minimally invasive manner. This avoids reaching the neurovascular-rich deep stroma while directly delivering drugs to the target area, significantly improving delivery efficiency and achieving sustained release. Nanosilver (AgNPs) exert their antibacterial effects through multiple mechanisms, including direct interaction with sulfur proteins on the bacterial cell membrane surface to disrupt the cell membrane, continuous release of silver ions to interfere with microbial metabolism, and catalytic generation of reactive oxygen species to induce oxidative damage. Therefore, it possesses advantages such as high efficiency, broad-spectrum antibacterial activity, low biotoxicity, and good stability, effectively inhibiting various bacteria, fungi, and viruses without easily inducing drug resistance.

[0004] To date, researchers have explored various materials and configurations for the construction of corneal microneedles. For example, while pure polymer microneedles (such as polylactic-co-glycolic acid copolymer PLGA and hyaluronic acid) exhibit good biocompatibility, their mechanical properties are often negatively correlated with drug loading and degradation rate, and they have limited functionality. To address the issue of controlled drug release, some studies have employed microneedles loaded with nanoparticles; however, their fabrication processes are complex, and burst drug release remains difficult to avoid. Recently, research has also focused on the morphological design of microneedles, such as hollow microneedles for liquid delivery, but their structure is fragile and cannot achieve the sequential release of multiple drugs.

[0005] Despite progress in improving delivery efficiency, most current corneal microneedle designs still have significant limitations, making it difficult to meet the clinical treatment needs of complex conditions such as infectious keratitis. Specifically: First, the drug delivery mode is singular and the release is uncontrollable. Existing microneedles mostly adopt the "overall mixed drug delivery" or surface coating mode, and the drug release depends on the overall degradation or rapid dissolution of the material, exhibiting a transient "burst release" characteristic. This mode cannot simulate the sequential drug delivery logic of "rapid anti-infection first, followed by sustained anti-inflammatory" required in clinical treatment, resulting in the inability to maintain effective concentrations after the critical treatment window.

[0006] Second, it lacks the ability to dynamically regulate the treatment process. As a disposable drug delivery device, the function of traditional microneedles is fixed at the moment of implantation and cannot respond to changes in the microenvironment during treatment (such as a decrease in pH, an increase in enzyme activity, or the emergence of drug-resistant bacteria). When it is necessary to adjust the treatment plan, it can only rely on re-administering the drug or replacing the device, and cannot achieve intelligent and adaptive treatment.

[0007] Third, there is a lack of synergistic therapy and functional integration. Infectious inflammation is often accompanied by complex biofilm formation, excessive inflammatory response, and tissue repair impairment. Existing microneedle designs mostly focus on single drug delivery and lack the ability to effectively integrate and spatially arrange multiple functional modules such as antibacterial (e.g., antibiotics), anti-inflammatory (e.g., glucocorticoids), and repair-promoting (e.g., growth factors), making it difficult to achieve synergistic effects.

[0008] Fourth, there is the challenge of balancing mechanical performance and safety. To carry sufficient drug, microneedles often need to be larger or made of different materials, which may weaken their mechanical strength, leading to incomplete puncture or breakage; while overly rigid materials may increase the risk of accidental damage to the deep layers of the cornea. How to achieve high drug loading and multifunctionality while ensuring safe penetration is a key contradiction.

[0009] Therefore, there is an urgent need in this field for a novel corneal drug delivery system that can not only efficiently penetrate the corneal barrier but also serve as a treatment platform, achieving multifunctional integration such as "time-sequential drug release," "dynamic treatment response," and "synergistic anti-inflammatory and antibacterial effects." Through innovative structural design, the entire process from rapid and potent treatment to long-term stable regulation can be completed in a single minimally invasive application, thereby fundamentally improving the treatment efficacy for infectious corneal inflammation. Summary of the Invention

[0010] The technical problem to be solved by the present invention is to overcome the defects in the prior art, thereby providing a concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes, its preparation method and application.

[0011] To achieve the above objectives, the present invention adopts the following technical solution: A concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes, comprising: Concave transparent substrate; A microneedle array is formed on the concave surface of the concave transparent substrate; The microneedle array includes several microneedle structures, and the microneedle structures include: Needle body; A needle tip is located at the top of the needle body. The needle tip is composed of a soluble polymer material and a photoresponsive composite. The photoresponsive composite is used to generate photothermal and / or photodynamic effects under near-infrared light irradiation. The photoresponsive complex comprises hydroxyl-functionalized graphyne and silver nanoparticles loaded on hydroxyl-functionalized graphyne.

[0012] In the above scheme, the concave transparent substrate has a concave structure that matches the physiological curvature of the human cornea, allowing the microneedle array to closely adhere to the corneal surface, solving the problems of poor conformability and easy slippage of traditional planar microneedles. The microneedle array penetrates the corneal epithelial barrier in a minimally invasive manner, directly delivering the active ingredients in the needle tip to the target tissue layer, significantly improving drug bioavailability. The needle tip is composed of a soluble polymer material and a photoresponsive complex. The soluble polymer material serves as a matrix that dissolves in the corneal tissue fluid, releasing the active ingredients. The hydroxyl-functionalized graphylene in the photoresponsive complex has anti-inflammatory activity, and the silver nanoparticles loaded on it have broad-spectrum antibacterial effects. The two work synergistically to achieve dual functions of anti-inflammatory and antibacterial properties. At the same time, the photoresponsive complex can generate photothermal and / or photodynamic effects under near-infrared light irradiation. By controlling the light conditions, the drug release rate and treatment intensity can be externally adjusted as needed, achieving dynamic and controllable treatment.

[0013] Preferably, the concave transparent substrate is made of hyaluronic acid, and the radius of curvature of the concave transparent substrate is 7.8mm-8.4mm, which precisely matches the curvature of the human cornea to ensure adhesion stability. The light transmittance of the concave transparent substrate is greater than or equal to 90% at visible light wavelength, which facilitates observation of the corneal condition through devices such as slit lamps during treatment. The soluble polymer material of the needle tip is hyaluronic acid, and the molecular weight of the soluble polymer material is 10. 4 g / mol-10 6 It has g / mol and good biocompatibility and degradability.

[0014] Preferably, the mass ratio of the photoresponsive composite to the soluble polymer material is 1:8 to 1:3; The loading of silver nanoparticles on the hydroxyl-functionalized graphyne is 1.0wt%-5.0wt%. This ratio range ensures that the needle tip has sufficient mechanical strength to puncture the corneal epithelium, while also releasing an effective therapeutic concentration of active ingredient after dissolution.

[0015] Preferably, the total height of the microneedle structure is 200μm-400μm; The microneedle structure has a base diameter of 30μm-60μm. This size design ensures that the microneedles can penetrate the corneal epithelium while avoiding contact with the deep stroma rich in nerves and blood vessels, thus achieving minimally invasive and painless treatment.

[0016] Preferably, the elastic modulus of the microneedle structure in the axial loading direction is 0.8 GPa-1.5 GPa; The maximum breaking load of the microneedle structure under axial compression is not less than 0.2N. This ensures that the microneedle does not break or bend during puncture, guaranteeing treatment safety.

[0017] Preferably, the complete dissolution time of the needle tip in the corneal tissue fluid environment is 25-30 minutes. This dissolution time enables rapid initial drug release, meeting the treatment needs of early and potent anti-infection in infectious keratitis. Subsequently, the entire microneedle and its substrate naturally and safely dissolve and metabolize completely in the ocular surface environment, without the need for secondary removal.

[0018] The above-mentioned method for preparing concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes includes the following steps: Hydroxyfunctionalized graphyne was dispersed in a silver salt solution, and a reducing agent was added to carry out an in-situ reduction reaction to obtain a photoresponsive complex loaded with silver nanoparticles. The complex was dissolved in a solvent with hyaluronic acid in a certain proportion to form a uniform needle tip slurry; Hyaluronic acid solution is poured into a concave mold with corneal curvature and freeze-dried to form a concave transparent substrate; The needle tip slurry is filled into the needle tip cavity of the microneedle mold, and then centrifuged and freeze-dried to form a microneedle array; The concave transparent substrate is bonded to the microneedle array, dried, and demolded to obtain a microneedle-substrate composite.

[0019] In the above scheme, the preparation method adopts a modular process route, which independently performs the synthesis of the photoresponsive complex, the preparation of the needle tip slurry, the molding of the concave substrate, and the molding of the microneedle array. Finally, the modules are integrated into one unit through a bonding step. This process design allows each functional unit to be independently optimized and replaced, facilitating flexible adjustment of material ratios or active ingredients according to different treatment needs, and has high scalability. At the same time, the use of freeze-drying molding process avoids the damage of active ingredients by high temperature or organic solvents, maintaining the structural integrity and functional activity of the hydroxyl-functionalized graphyne and silver nanoparticles.

[0020] Preferably, the silver salt solution is a silver nitrate solution with a concentration of 5mM-20mM, and the mass ratio of the hydroxyl-functionalized graphyne to the silver nitrate solution is 1:1-1:5. This concentration and ratio range can ensure that the silver nanoparticles are uniformly loaded on the graphyne sheets, avoid particle aggregation, and ensure the stable performance of photothermal and photodynamic effects.

[0021] The reducing agent is sodium ascorbate. The in-situ reduction reaction is carried out at room temperature for 2-6 hours. Sodium ascorbate is used as a mild reducing agent, and the in-situ reduction of silver ions can be achieved at room temperature without the need for high temperature or strong reducing agent. The reaction conditions are mild and controllable, which is beneficial to maintaining the integrity of the graphyne sheet structure and surface functional groups.

[0022] The freeze-drying conditions are -50°C and below 10 Pa for 24-48 hours.

[0023] Preferably, the preparation method further includes: Terminal sterilization was performed using cobalt-60 gamma irradiation at doses of 20kGy-30kGy.

[0024] The above-mentioned concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes, or the concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes prepared by the above-mentioned method, are used in the preparation of medical devices for the treatment or adjuvant treatment of eye diseases. The eye disease mentioned is selected from bacterial keratitis, post-corneal infection, corneal inflammation, or corneal epithelial defects.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This application achieves painless and minimally invasive high-efficiency drug delivery. It utilizes a high-density microneedle array mounted on a hyaluronic acid concave substrate with the physiological curvature of the cornea. The microneedles instantly physically puncture the corneal epithelial barrier (penetration depth 100-200 μm) to directly deliver therapeutic drugs to the target tissue layer. This method overcomes the limitation of traditional eye drops having a bioavailability of less than 5%, while avoiding the pain, bleeding, and infection risks associated with invasive procedures such as subconjunctival injections, achieving a balance between high-efficiency treatment and minimally invasive procedures.

[0026] 2. This application innovatively integrates a synergistic anti-inflammatory and antibacterial therapeutic function at the microneedle tip, combining a "hydroxyl-functionalized graphylene-supported nano-silver" composite material. Firstly, utilizing the strong reducing properties and abundant specific surface area of ​​GDYO, the precursor GDYO-Ag is synthesized in situ in a green manner, with uniformly distributed AgNPs on its surface, preventing aggregation. Secondly, the addition of the positively charged modifier DAPT during the synthesis of GDYO-Ag not only regulates the synthesis of small-sized AgNPs but also alters the electronegativity of GDYO, enhancing its adsorption to bacteria and thus strengthening its antibacterial properties. Thirdly, the synthesized GDYO-Ag complex can promote photocatalysis to generate more ROS through photothermal properties, improving bactericidal performance while maintaining good biocompatibility and preventing drug resistance. Nano-silver provides potent, broad-spectrum, immediate antibacterial activity, while hydroxyl-functionalized graphylene itself possesses excellent anti-inflammatory activity, inhibiting key inflammatory factors. Both drugs are released synergistically at the lesion site, forming a dual-targeted treatment strategy of "antibacterial-anti-inflammatory" to simultaneously address pathogen clearance and control of excessive inflammatory response in infectious keratitis, thus solving the problem of limited efficacy of single-drug therapy.

[0027] 3. Achieving precise timing of treatment and safe in vivo metabolism, this application adopts a "fully soluble" design concept, possessing precise timing control capabilities. The needle tip area dissolves rapidly within 30 minutes, achieving an initial high concentration release of the therapeutic drug; subsequently, the entire microneedle and hyaluronic acid base, after fulfilling their missions of mechanical support and drug delivery, naturally and safely dissolve and metabolize completely in the ocular surface environment, without the need for secondary surgery to remove it, thus completely avoiding the risks of chronic inflammation, corneal neovascularization, or rejection that may be caused by long-term retention of non-degradable materials.

[0028] 4. A scalable modular drug delivery platform has been established. The structural design of this application features highly modular functionality. The hyaluronic acid substrate, microneedle body, and functionalized needle tip region can be optimized and replaced as independent modules. In the future, the drugs or active factors loaded on the needle tip (such as cyclosporine A, anti-VEGF antibodies, antifungal drugs, etc.) can be flexibly changed according to different disease needs (such as dry eye, corneal neovascularization, fungal infections, etc.), enabling the rapid development of a series of products for specific indications, demonstrating strong platform scalability and clinical applicability prospects. Attached Figure Description

[0029] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of the overall structure of the concave light-controlled microneedle provided in this embodiment.

[0031] Figure 2 This is a schematic diagram of the microneedle array acting on the ocular surface in this embodiment.

[0032] Figure 3 This is a schematic diagram of the dissolution of the microneedle array in this embodiment.

[0033] Figure 4 This is a schematic diagram of the overall structure of the microneedle structure provided in this embodiment.

[0034] Explanation of reference numerals in the attached figures: 1. Concave transparent substrate; 2. Microneedle structure. Detailed Implementation

[0035] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the raw materials, reagents, or apparatus used in the examples, comparative examples, and test examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0037] Example 1 A method for preparing concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes, the specific preparation method is as follows: Step 1: Disperse hydroxyl-functionalized graphyne in a silver salt solution, add a reducing agent to carry out an in-situ reduction reaction, and obtain a photoresponsive complex loaded with silver nanoparticles. Step 2: Dissolve the complex and hyaluronic acid in a solvent in a certain proportion to form a uniform needle tip slurry; Step 3: Pour the hyaluronic acid solution into a concave mold with corneal curvature, and freeze-dry it to form a concave transparent substrate 1; Step 4: Fill the needle tip slurry into the needle tip cavity of the microneedle mold, and then centrifuge and freeze-dry to form a microneedle array; Step 5: Adhere the concave transparent substrate 1 to the microneedle array, dry it, and demold it to obtain the microneedle-substrate composite. Step 6: Terminal sterilization is performed by irradiating with cobalt-60 gamma rays at a dose of 20kGy-30kGy.

[0038] It should be understood that step one specifically includes the preparation of graphyne oxide and the preparation of the photoresponsive composite.

[0039] Specifically, regarding the synthesis of hydroxyl-functionalized GDYO (graphyne oxide).

[0040] The synthesis of GDY (graphyne) was prioritized. Copper sheets were washed sequentially with hydrochloric acid solution (4M), ultrapure water, and ethanol under ultrasonic cleaning. The copper sheets and 60 mL of pyridine were then added to a three-necked flask and heated at 120 °C for 1 hour under nitrogen protection. Next, 200 mg of hexaethynylbenzene monomer (HEB) was dissolved in tetrahydrofuran (THF) and stirred in a liquid nitrogen bath for 30 minutes. 2.5 mL of a THF solution containing tetrabutylammonium fluoride was added to the system, and stirring was continued at low temperature for 15 minutes. The reaction mixture was diluted with ethyl acetate, washed three times with saturated sodium chloride solution, dried, and filtered to obtain the dried HEB precursor. This precursor was dissolved in 50 mL of pyridine and transferred to a nitrogen-protected constant-pressure funnel. The solution was slowly added dropwise over 10 hours at 80 °C to a mixture containing pyridine (60 mL) and copper sheets. The reaction was carried out at 120 °C for 3 days. After the reaction, the material was freeze-dried to remove pyridine. The solid product formed on the surface was collected by centrifugation and washed successively with hot N,N-dimethylformamide (80°C) and ethanol (70°C), and then dried to obtain GDY powder. Finally, the GDY powder was dispersed in ultrapure water and ultrasonically treated at 750 W and 25°C for 10 hours to obtain GDY nanosheets.

[0041] Next, the oxidation of GDY was performed. 500 mg of graphyne was fully dispersed in 50 ml of a 1% sodium hypochlorite solution to achieve a graphyne concentration of 10 mg / ml. Because graphyne lacks oxidizing groups, the prepared mixture showed significant precipitation. The mixture was pre-treated with an ultrasonic cleaner for 30 minutes to fully disperse the graphyne powder. The mixture was stirred uniformly at 25°C in the dark. After 6 hours, the oxidation reaction was stopped by heating to 100°C for 5 minutes. After cooling to room temperature, the mixture was centrifuged at 10,000 rpm for 10 minutes and washed 2-3 times with 200 ml of distilled water to remove residual sodium hypochlorite solution. The material was dialyzed in a dialysis bag for 3 days to remove any possible oxidizing agents and metal ion impurities. After drying and pulverizing, the oxidized graphyne powder was placed in 1 ml of ultrapure water and sonicated for 10 minutes to obtain a dispersion of oxidized graphyne.

[0042] Specifically, regarding the synthesis of photoresponsive complexes.

[0043] Prepare a dispersion of 100 μg / ml graphyne oxide in 500 ml of water. Add 100 mg of silver nitrate powder, stir thoroughly, and sonicate for 30 minutes. Heat to boiling under stirring to ensure uniform dispersion of silver ions. After boiling for 30 minutes, stir and cool. Prepare a reducing solution by adding 10 mg of ascorbic acid to 10 ml of ultrapure water to a concentration of 1 mg / ml. When the mixture of graphyne oxide and silver nitrate cools to below 25°C, gradually add the ascorbic acid reducing solution dropwise. Stop adding the solution when the mixture changes from yellowish-brown to dark brown, and continue stirring for 6 hours. Let stand overnight. Filter and wash the precipitated material in the mixture thoroughly, then freeze-dry in a freeze dryer. The collected powder is the Ag@GDYO nanocomposite (i.e., the photoresponsive composite).

[0044] Step two specifically includes the preparation of needle tip slurry.

[0045] Specifically, 0.2 g of the graphdiyne-silver nanocomposite powder prepared in the above steps was weighed out, along with 0.8 g of hyaluronic acid (HA, molecular weight approximately 10). 5 Mix (g / mol). Slowly add the mixture to 10 mL of deionized water and homogenize using a high-speed homogenizer at 10,000 rpm for 15 minutes under ice-water bath conditions to form a uniform, dispersed composite slurry. In this slurry, the mass ratio of hydroxygraphyne to HA is 1:4, which is used to form the tip region of the microneedles to ensure rapid dissolution and release of the active ingredient in the corneal tissue fluid.

[0046] Step three specifically involves the preparation of the concave transparent substrate 1.

[0047] Specifically, hyaluronic acid is dissolved in deionized water to prepare a 10% (w / v) hyaluronic acid solution. This solution is filtered, degassed, and allowed to stand for later use. It is then used to prepare a concave transparent substrate 1 with corneal-adaptive curvature. This substrate is formed by freeze-drying using an independent mold. The radius of curvature of the concave transparent substrate is 7.8-8.4 mm, its center thickness is 80-150 μm, its edge thickness is 200-300 μm, and its light transmittance at visible light wavelengths is ≥90%. Preferably, the radius of curvature is 8.0 mm, and the center thickness is 100 μm.

[0048] Step four specifically involves the fabrication of the microneedle array.

[0049] Specifically, a custom-designed PDMS microneedle mold (with a conical cavity, 250 μm depth, and 50 μm bottom diameter) is used, and the needle tip composite slurry prepared in step two is evenly coated onto the mold surface. The mold is then placed in a centrifuge and centrifuged at 5000 r·min. -1Centrifuge for 30 minutes to ensure the slurry completely fills the deepest part of the microneedle tip cavity and removes air bubbles. After centrifugation, scrape off any excess slurry from the mold surface. Pre-freeze the filled mold at -20°C for 2 hours, then transfer it to a freeze dryer and freeze-dry at -50°C and a vacuum of less than 10 Pa for 24 hours to completely solidify the tip area.

[0050] Step five specifically involves the preparation of the microneedle-substrate complex.

[0051] Specifically, a small amount of the concave substrate solution prepared in step three is poured onto the surface of the dried microneedle mold from step four, forming a thin "adhesive layer." A pre-prepared independent concave transparent substrate (freeze-dried using an independent mold, with a curvature radius of 8.0 mm and a thickness of 100 μm) is gently placed over the mold coated with the adhesive layer, and a slight, uniform pressure (approximately 0.5 N / cm²) is applied to ensure a tight bond between the substrate and the roots of the microneedle array. The bonded mold is then placed in a freeze dryer for 12 hours to allow the interface layer to fully solidify and fuse. Finally, under room temperature and dry conditions, the complete microneedle-substrate composite is gently peeled from the PDMS mold to obtain light-controlled microneedles with a concave structure.

[0052] Step six involves vacuum packaging the prepared photocontrolled microneedles and sterilizing them by cobalt-60 gamma irradiation at a dose of 25 kGy to ensure the product is sterile and maintain the activity of the material.

[0053] The concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes, prepared according to the above method, include a concave transparent substrate 1 and a microneedle array, wherein the microneedle array is formed on the concave surface of the concave transparent substrate 1. The microneedle array includes several microneedle structures 2, each including a needle body and a needle tip. The needle tip is located at the top of the needle body and is composed of a soluble polymer material and a photoresponsive complex. The photoresponsive complex is used to generate photothermal and / or photodynamic effects under near-infrared light irradiation. The photoresponsive complex includes hydroxyl-functionalized graphyne and silver nanoparticles loaded on hydroxyl-functionalized graphyne.

[0054] Specifically, the concave transparent substrate 1 is made of hyaluronic acid and has a concave structure that matches the physiological curvature of the human cornea, with a radius of curvature of 7.8mm-8.4mm and a transmittance of greater than or equal to 90% at visible light wavelengths. A microneedle array is formed on the concave surface of this substrate, comprising several microneedle structures 2. Each microneedle structure 2 includes a needle body and a needle tip at the top of the needle body. The needle tip is made of a soluble polymer material (hyaluronic acid, molecular weight 10). 4 g / mol-10 6The drug is composed of a photosensitive compound (g / mol) and a photoresponsive complex. The photoresponsive complex includes hydroxylated functionalized graphyne (preferably hydroxylated flake graphyne) and silver nanoparticles loaded thereon (loaded onto hydroxylated functionalized graphyne via in-situ reduction). This complex can generate photothermal and / or photodynamic effects under near-infrared light irradiation, achieving photocontrolled drug release and synergistic anti-inflammatory and antibacterial effects.

[0055] Furthermore, the total height of the microneedle structure is 200μm-400μm, and the base diameter is 30μm-60μm, enabling it to penetrate the corneal epithelium (approximately 50-100μm thick) while avoiding contact with the deep stroma rich in nerves and blood vessels. The elastic modulus of the microneedle structure in the axial loading direction is 0.8GPa-1.5GPa, and the maximum breaking load of a single needle under axial compression is not less than 0.2N. The mass ratio of the photoresponsive complex to the soluble polymer material (hyaluronic acid) in the needle tip is 1:8-1:3, and the loading of hydroxylated functionalized graphylene nanoparticles is 1.0wt%-5.0wt%, with a particle size of 10nm-30nm. The needle tip completely dissolves in the corneal tissue fluid environment in 25-30 minutes, enabling rapid drug release in the early stages of treatment. Subsequently, the entire microneedle and substrate gradually and safely degrade in the ocular surface environment, eliminating the need for secondary surgical removal.

[0056] Experimental Example 1 Solubility behavior characterization test The specific steps for performing an in vitro dissolution experiment include: A group of scaffold samples (n=5) were accurately weighed (initial mass W0) and immersed in 5 mL of PBS solution (pH 7.4, 37℃) containing collagenase I (10 U / mL). The degradation solution was replaced with fresh solution every 3 days to maintain enzyme activity. One sample was removed weekly, carefully rinsed with ultrapure water, freeze-dried, and accurately weighed again (post-degradation mass W). t ). Calculate the mass residual rate: MassResidual(%) = (W t / W0)×100%.

[0057] The experimental results showed that the residual mass of the stent was approximately 20% at week 8 (day 56), indicating that 80% of it had degraded. Its molecular weight decreased to approximately 15% of its initial value at week 8. These results demonstrate that the microneedle possesses excellent degradation characteristics under physiological conditions, confirming that its soluble design enables safe metabolism on the ocular surface and ensures long-term biocompatibility.

[0058] Experimental Example 2 Mechanical property testing The specific steps for conducting a tensile test include: A set of substrate samples (n=5) before degradation (week 0) were made into standard dumbbell-shaped specimens and subjected to tensile testing at room temperature using a universal testing machine, according to ASTM D638 standard, with a tensile rate of 10 mm / min.

[0059] The experimental results showed that the average elastic modulus was 1.52 ± 0.15 MPa and the elongation at break was 60 ± 5%. These results indicate that the substrate material has moderate mechanical strength, good ductility, and excellent flexibility, which can meet the mechanical conformal requirements for fitting the curvature of the cornea.

[0060] Experimental Example 3 Puncture capability test The microneedle puncture performance was evaluated using porcine skin tissue, and the specific steps included: Microneedles were vertically inserted into pig skin samples and held for 1 minute before separation. After removing the light-controlled microneedles, the puncture area was photographed, and the pig skin samples were soaked in tissue fixative for 3 hours. The treated skin was then embedded in paraffin to prepare 5 μm thick sections, which were stained with hematoxylin and eosin (H&E). Finally, the images were recorded using an optical microscope.

[0061] The experimental results were as follows: Microneedles were pressed into the pigskin and held for 1 minute before being removed. After removal, a complete 15×15 micropore array was observed on the skin surface. Histological examination of the pigskin model using H&E staining showed an average puncture depth of approximately 120 μm. Due to the viscoelasticity of the skin, the puncture depth was less than the length of the microneedle.

[0062] Test Example 4 In vitro light-controlled antibacterial activity test The specific steps for preparing GDYO-Ag solution and conducting antibacterial experiments include: Prepare a GDYO-Ag solution with a concentration of 1 mg / mL. Take 100 μL of GDYO-Ag solution into a 1.5 mL centrifuge tube and add 800 μL of sterile water. Add 2 × 10⁻⁶ mg / mL of water to the above solution. 8 The CFU / mL model bacterial suspension was 100 μL, the final sample concentration was 100 μg / mL, and the bacterial concentration was 2 × 10⁻⁶. 7 CFU / mL. After 30 min of UV irradiation or 30 min of light-protected exposure, serially dilute to a concentration of 10. 4 10² CFU / mL, spread evenly on a solid culture medium plate, and after the liquid has dried, invert it and incubate in a 37°C incubator for 12 or 24 hours. Each experiment was repeated three times.

[0063] The experimental results are as follows: The GDYO-Ag light-shielded group exhibited moderate antibacterial effect after 30 minutes of contact with bacteria solely through the material itself, with an average sterilization rate of 61.4%. This indicates that silver ions in the GDYO-Ag composite material were continuously released, exerting a toxic effect on bacteria. However, due to the lack of external energy excitation, the photothermal / photodynamic effect of graphdiene oxide was not activated, thus failing to achieve complete sterilization. The GDYO-Ag ultraviolet irradiation group exhibited extremely strong antibacterial activity after 30 minutes of contact with ultraviolet light, with an average sterilization rate exceeding 99.9%.

[0064] Experimental Example 5 Photocatalytic performance test The specific steps for ROS detection using DCFH as a fluorescent probe include: The fluorescence intensity of DCF under visible light irradiation was measured using a fluorescence spectrometer. Test subjects included HA, HA-GDYO, and HA-GDYO-Ag. 0.5 mL of DCFH-DA solution (1 mM) was mixed with 2.0 mL of NaOH solution (50 mM) and incubated in the dark for 30 min to activate DCFH-DA. The activated DCFH-DA was then added to 10 mL of PBS (pH=7.4, 25 mM) for later use. 50 μL of DCFH-DA solution was added to 2.0 mL of HA, HA-GDYO, and HA-GDYO-Ag (10 ppm) solutions, respectively. The control group was replaced with 2 mL of PBS. Fluorescence spectroscopy analysis was performed by sampling at 2-min intervals during VL irradiation, with continuous irradiation for 30 min.

[0065] The experimental results are as follows: When HA-GDYO-Ag was added to a methyl orange solution and the mixture was irradiated with VL (volatile liquid volume), the characteristic absorption peak of MO at 464 nm decreased with increasing irradiation time. Using a blank methyl orange solution at 464 nm under VL irradiation as a control, it is speculated that the photodegradation of MO is due to the complexation of GDYO and Ag, which promotes photoelectric separation efficiency and increases ROS generation. ROS was evaluated using the DCFH-DA fluorescent probe under VL irradiation. With increasing irradiation time, the fluorescence intensity of DCFH-DA from HA-GDYO and HA-GDY-Ag gradually increased, while the fluorescence intensity of pure DCFH-DA under the same conditions showed almost no increase. These results indicate that more ROS are generated in the presence of HA-GDYO and HA-GDYO-Ag.

[0066] Based on the above test examples 1 to 5, the following conclusions can be drawn: PCT+PTT combined therapy rapidly kills MRSA through photothermal effects, while reducing inflammation and promoting tissue repair through photochemical effects. It is superior to single photodynamic therapy or photothermal therapy in reducing corneal opacity, inhibiting neovascularization, reducing inflammatory infiltration, and promoting epithelial healing.

[0067] Experimental Example 6 Biological validation of light-controlled microneedles in a keratitis model includes the following steps: Step 1: Establishment of an animal model All animals had free access to food and water, and the ambient temperature was maintained at 20-26℃ with a relative humidity of 40%-60%. Male Sprague-Dawley rats (SD rats), weighing approximately 180-200g, were in good health with transparent corneas. After a week of acclimatization, the SD rats were intraperitoneally anesthetized with a standard dose of 10% chloral hydrate solution. After anesthesia, a 3mm diameter central wound was created by scraping away the cornea of ​​the right eye; the left eye was left untreated. Subsequently, 5μL of MRSA bacterial suspension at a concentration of 10⁸ CFU·mL⁻¹ was inoculated into the wound in the right eye to establish the model. After successful modeling, the animals were randomly divided into 4 groups of 6 animals each, and microneedles were first placed in the right eye for different interventions. Step 2: Grouping Control group: No intervention was performed after modeling; Photodynamic therapy group (PCT group): After modeling, the light-controlled microneedles prepared in Example 1 were placed in the right eye and irradiated with visible light (VL); Photothermal therapy group (PTT group): After modeling, the light-controlled microneedles prepared in Example 1 were placed in the right eye and irradiated with 808nm near-infrared light (NIR); Combined treatment group (PCT+PTT group): After modeling, the light-controlled microneedles prepared in Example 1 were placed in the right eye and irradiated with visible light combined with 808nm near-infrared light (VL+808nmNIR); MnO x / GDY microneedle kit: After modeling, MnO prepared according to the method disclosed in Chinese Patent CN118045034A was placed in the right eye. x / GDY microneedles (MGMN) do not use near-infrared light, based on their description of "no need for near-infrared light assistance".

[0068] It should be noted that setting MnO x The purpose of the / GDY microneedle assembly is to address the shortcomings of existing technologies, such as the Chinese patent CN118045034A, which uses MnO. xUsing GDY as the functional material, this invention employs a photoresponsive complex (GDYO-Ag) formed by combining hydroxyl-functionalized GDYO with in-situ reduced nanosilver. By limiting the control group to not applying near-infrared light, the inherent differences in antibacterial and anti-inflammatory properties of the two materials on the corneal microneedle platform were compared independently. The same treatment was administered to the right eye of rats under different conditions every other day, and observation, scoring, and photographic recording were performed using a slit lamp. Subsequently, the experimental animals were euthanized, and eyeballs were collected for histopathological and immunofluorescence analysis to evaluate the treatment effect.

[0069] Evaluation of the effectiveness of the above experiments (1) Slit-lamp observation: Healthy group: clear corneas, no epithelial defects, no neovascularization, and negative fluorescein staining.

[0070] Control group (untreated infection): On day 1 post-infection, obvious grayish-white infiltrates, approximately 3-4 mm in diameter, are visible on the cornea, accompanied by severe edema. On days 3-5, corneal ulceration worsens, with copious purulent discharge; fluorescein staining reveals extensive epithelial defects. On day 7, corneal opacity (occultation score 3-4) develops, with extensive neovascularization; corneal perforation or symblepharon may occur in some eyes.

[0071] PCT group (VL group): On day 3 post-infection, corneal edema and infiltration were slightly milder than in the Control group. On day 7, the cornea still had obvious corneal scars (cloudiness score 2-3), more neovascularization, and the epithelium was basically healed but the healing speed was slower.

[0072] PTT group (808nm NIR group): Due to the simple photothermal effect of sterilization, the bacterial load decreased rapidly in the early stage (days 1-3). From day 5 to 7, corneal edema subsided significantly, but the thermal effect may cause slight collateral damage, resulting in mild opacity of the corneal stroma (opause score 1-2) and a small amount of neovascularization.

[0073] PCT+PTT group (combined treatment group): Days 1-3: Corneal edema and infiltration subsided rapidly, and the ulcer surface shrank significantly.

[0074] Day 5: The corneal epithelium has basically healed, and only punctate staining is observed with sodium fluorescein staining. Day 7: The cornea has returned to transparency or only has mild cloudy opacity (opause score 0-1), and no obvious neovascularization is observed. The ocular surface structure is well preserved, significantly better than other treatment groups.

[0075] (2) Histological analysis: Control group: The corneal epithelial structure was completely destroyed or missing, the stromal layer was severely edematous, a large number of neutrophils were diffusely infiltrated (full field of view), corneal stromal cells (fibroblasts) were necrotic or missing, and the corneal thickness was significantly increased.

[0076] PCT group: Partial but incomplete regeneration of the epithelial layer, with a large number of inflammatory cells still infiltrating the stroma layer, and a small number of neovascular lumens visible.

[0077] PTT group: The epithelial layer was basically intact, and the infiltration of inflammatory cells in the stroma layer was significantly reduced, but the structure of the stroma layer was slightly disordered, and vacuolation of stromal cells could be seen in some areas (mild damage from thermal effects).

[0078] PCT+PTT group: The corneal epithelium is intact and continuous (about 5-6 layers of cells), the stromal layer has a dense structure and regular arrangement, almost no inflammatory cell infiltration is seen, the corneal thickness is close to normal, and neovascularization is rare.

[0079] (3) Detection of inflammatory factors Control group: The infection peaked on days 3-5 post-infection, reaching extremely high levels.

[0080] PCT group: The level is slightly lower than that of the Control group, but still at a high level.

[0081] PTT group: levels were significantly reduced because photothermal action rapidly eliminated bacteria, reducing the continuous stimulation of bacterial endotoxins and metabolites.

[0082] The PCT+PTT group had the lowest levels (significantly lower than the PCT and PTT groups, p<0.01), indicating that the combined treatment not only cleared the bacteria but also may have modulated the immune response through photochemical effects, preventing excessive inflammation.

[0083] (4) with MnO x Comparison of GDY microneedle groups Table 1. This invention and MnO x Comparison of GDY microneedle groups

[0084] From the experimental data in Table 1, we can see that MnO x / GDY microneedle group depends on MnO x While the four enzymes in the traditional method have a continuous biochemical reaction, they lack an "explosive" pharmacological response during the outbreak phase of infection. However, this invention achieves precise intervention by utilizing the dual photothermal and photodynamic effects of GDYO-Ag, enabling "light to reach the drug" and more rapidly suppressing pathogens during the critical treatment window (first 3 days), thereby producing significant differences in the final healing quality, epithelial thickness, and transparency (p<0.01).

[0085] Therefore, based on the innovation of functional materials, this invention further introduces the photothermal / photodynamic effect of near-infrared light response, realizing the external dynamic regulation of drug release. By comparing the control group (without light control) with the PCT+PTT combined treatment group of this invention (with light control), the incremental therapeutic effect brought about by the light-controlled release dimension is verified.

[0086] The above head-to-head comparison demonstrates that the advantages of the technical solution of this invention in terms of corneal transparency restoration, epithelial healing speed, inflammation suppression, and neovascularization control are not simply a superposition of graphdiyne microneedle technology and known photothermal release mechanisms, but rather an unexpected technical effect produced by the synergy of "specific GDYO-Ag composite material + microneedle tip positioning + near-infrared photocontrolled graded release".

[0087] Experimental Results: The composite concave light-controlled microneedle provided by this invention, by organically combining corneal curvature adaptation design, soluble polymer materials and graphyne-silver nanophotosensitive materials, not only achieves non-invasive and precise drug delivery, but also significantly enhances the antibacterial and anti-inflammatory effects through light-controlled synergistic effects. It has extremely high clinical application value in the treatment of bacterial keratitis, post-corneal surgery anti-infection and promotion of corneal repair.

[0088] The above embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art based on the present invention shall fall within the scope of protection claimed by the present invention.

Claims

1. A concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes, characterized in that, include: Concave transparent substrate; A microneedle array is formed on the concave surface of the concave transparent substrate; The microneedle array includes several microneedle structures, and the microneedle structures include: Needle body; A needle tip is located at the top of the needle body. The needle tip is composed of a soluble polymer material and a photoresponsive composite. The photoresponsive composite is used to generate photothermal and / or photodynamic effects under near-infrared light irradiation. The photoresponsive composite includes graphyne oxide and silver nanoparticles supported on graphyne oxide. The concave transparent substrate is made of hyaluronic acid, and the radius of curvature of the concave transparent substrate is 7.8mm-8.4mm; The soluble polymer material of the needle tip is hyaluronic acid.

2. The concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes according to claim 1, characterized in that, The transmittance of the concave transparent substrate is greater than or equal to 90% at visible light wavelengths; The molecular weight of the soluble polymer material is 10. 4 g / mol-10 6 g / mol.

3. The concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes according to claim 1, characterized in that, The mass ratio of the photoresponsive composite to the soluble polymer material is 1:8 to 1:3; The loading of silver nanoparticles on the graphdiyne oxide is 1.0wt%-5.0wt%.

4. The concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes according to claim 1, characterized in that, The total height of the microneedle structure is 200μm-400μm; The bottom diameter of the microneedle structure is 30μm-60μm.

5. The concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes according to claim 1, characterized in that, The elastic modulus of the microneedle structure in the axial loading direction is 0.8 GPa-1.5 GPa; The maximum fracture load of the microneedle structure under axial compression is not less than 0.2N.

6. The concave light-controlled microneedle for corneal anti-inflammatory and antibacterial purposes according to claim 1, characterized in that, The complete dissolution time of the needle tip in the corneal tissue fluid environment is 25-30 minutes.

7. The method for preparing the concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes according to any one of claims 1-6, characterized in that, The preparation method includes: Graphdiyne oxide was dispersed in a silver salt solution, and a reducing agent was added to carry out an in-situ reduction reaction to obtain a photoresponsive composite loaded with silver nanoparticles. The complex was dissolved in a solvent with hyaluronic acid in a certain proportion to form a uniform needle tip slurry; Hyaluronic acid solution is poured into a concave mold with corneal curvature and freeze-dried to form a concave transparent substrate; The needle tip slurry is filled into the needle tip cavity of the microneedle mold, and then centrifuged and freeze-dried to form a microneedle array; The concave transparent substrate is bonded to the microneedle array, dried, and demolded to obtain a microneedle-substrate composite.

8. The preparation method according to claim 7, characterized in that, The silver salt solution is a silver nitrate solution with a concentration of 5mM-20mM, and the mass ratio of the graphdiyne oxide to the silver nitrate solution is 1:1-1:

5. The reducing agent is sodium ascorbate, and the in-situ reduction reaction is carried out at room temperature for 2-6 hours. The freeze-drying conditions are -50°C and below 10 Pa for 24-48 hours.

9. The preparation method according to claim 7, characterized in that, The preparation method further includes: Terminal sterilization was performed using cobalt-60 gamma irradiation at doses of 20kGy-30kGy.

10. The concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes as described in any one of claims 1-6, or the concave light-controlled microneedles for corneal anti-inflammatory and antibacterial purposes prepared by the preparation method described in any one of claims 7-9, are used in the preparation of medical devices for treating or adjuvant treatment of eye diseases; in, The eye diseases mentioned are selected from bacterial keratitis, post-corneal infection, corneal inflammation, or corneal epithelial defects.

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