A soluble microneedle patch for bacterial keratitis treatment and a preparation method and application thereof

Abstract

The present application relates to the technical field of biomedical materials, and particularly relates to a soluble microneedle patch for treating bacterial keratitis, a preparation method and application thereof. The soluble microneedle patch comprises a cross-linkable matrix, the cross-linkable matrix is obtained by acylation reaction of sodium chondroitin sulfate and methacrylic anhydride, and forms a three-dimensional cross-linked network; and chondroitin sulfate-selenium nanoparticles, the chondroitin sulfate-selenium nanoparticles form a dispersed phase in the three-dimensional cross-linked network formed by the cross-linkable matrix. The soluble microneedle patch has the advantages of excellent specific external dissolution performance, excellent biocompatibility, excellent antibacterial activity and excellent mechanical properties, can realize long-acting release of active ingredients, the dissolution rate of the soluble microneedle patch can be regulated by solid content, the soluble microneedle patch can effectively inhibit the growth of staphylococcus aureus, has sufficient mechanical strength, can minimally invasively penetrate the corneal barrier, realizes integrated safe and efficient treatment of bacterial keratitis, and has a good clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a soluble microneedle patch for the treatment of bacterial keratitis, its preparation method, and its application. Background Technology

[0002] Bacterial keratitis is a severe inflammatory disease of the cornea caused by pathogenic bacteria. It progresses rapidly and can easily lead to corneal ulcers, stromal resorption, and even perforation, making it a significant cause of vision loss. Antibiotics are commonly used clinically to treat bacterial keratitis, including fluoroquinolones, aminoglycosides, and vancomycin. However, prolonged or excessive use of these antibiotics can lead to increased bacterial resistance, reducing treatment effectiveness and making the treatment of bacterial keratitis more difficult. Therefore, there is an urgent need to develop new strategies for treating bacterial keratitis. In recent years, microneedles (MNs), as a novel drug delivery system, have shown promising application prospects in the treatment of ocular surface diseases due to their advantages such as being minimally invasive, painless, highly efficient in penetrating biological barriers, and capable of localized controlled release. However, existing microneedles suffer from problems such as poor biocompatibility, limited antibacterial effects, and insufficient mechanical properties, making it difficult to meet the treatment needs of bacterial keratitis.

[0003] Therefore, developing a novel functional microneedle with excellent biocompatibility, antibacterial activity, and mechanical properties is of great significance for constructing a treatment system for bacterial keratitis. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a soluble microneedle patch for the treatment of bacterial keratitis, its preparation method, and its application. This invention uses the product obtained from the acylation reaction of sodium chondroitin sulfate and methacrylic anhydride as a crosslinkable matrix, and chondroitin sulfate-selenium nanoparticles as the dispersed phase. The chondroitin sulfate-selenium nanoparticles are dispersed in a three-dimensional crosslinked network formed by the crosslinkable matrix to prepare a soluble microneedle patch. This soluble microneedle patch combines excellent in vitro dissolution performance, excellent biocompatibility, excellent antibacterial activity, and excellent mechanical properties. It can achieve long-term sustained release of active ingredients, and its dissolution rate can be controlled by the solid content. It can effectively inhibit the growth of Staphylococcus aureus and possesses sufficient mechanical strength (the single needle breaking force of the soluble microneedle patch in this embodiment is >0.5 N). It can minimally invasively penetrate the corneal barrier, achieving integrated, safe, and efficient treatment of bacterial keratitis, and has good clinical application prospects.

[0005] A first aspect of the present invention provides a soluble microneedle patch for the treatment of bacterial keratitis, the soluble microneedle patch comprising: A crosslinkable matrix, wherein the crosslinkable matrix is ​​photocrosslinked and cured to form a three-dimensional crosslinked network; Chondroitin sulfate-selenium nanoparticles, wherein the chondroitin sulfate-selenium nanoparticles form a dispersed phase in a three-dimensional cross-linked network formed by the cross-linkable matrix; The structural formula of the crosslinkable matrix is ​​as follows: .

[0006] The soluble microneedle patch for the treatment of bacterial keratitis, as described above, has a crosslinkable matrix and a chondroitin sulfate-selenium nanoparticle mass ratio of (5-10):1. And / or, the chondroitin sulfate-selenium nanoparticles have an average particle size of 120–130 nm, a polydispersity index (PDI) of 0.05–0.1, and a zeta potential of -41–-38 mV.

[0007] The soluble microneedle patch for the treatment of bacterial keratitis as described above is an integral soluble microneedle patch comprising a base and needle tips arranged in an array on the base; The needle tip is conical in shape, with a needle height of 590–610 μm and a needle base diameter of 290–310 μm.

[0008] A second aspect of the present invention provides a method for preparing the soluble microneedle patch for the treatment of bacterial keratitis, comprising the following steps: S1. Chondroitin sulfate sodium is dissolved in water and then methacrylic anhydride is added to carry out an acylation reaction. The product after the acylation reaction is subjected to a first dialysis treatment and a first freeze-drying treatment to obtain a loose and porous crosslinkable matrix. S2. Using sodium selenite as the selenium source, chondroitin sulfate as the stabilizer and capping agent, and L-ascorbic acid as the reducing agent, chondroitin sulfate-selenium nanoparticles are generated in situ through coordination, electrostatic adsorption and steric hindrance effect. S3. The crosslinkable matrix is ​​added to water to form a uniform and transparent crosslinkable matrix aqueous solution. The photoinitiator and the chondroitin sulfate-selenium nanoparticles are added to the crosslinkable matrix aqueous solution in sequence and then blended to obtain a microneedle precursor solution. S4. The microneedle precursor liquid is injected into the microneedle mold, and after degassing, photocrosslinking curing, and drying, the soluble microneedle patch is obtained.

[0009] In the preparation method of the soluble microneedle patch for the treatment of bacterial keratitis as described above, in step S1, the ratio of sodium chondroitin sulfate to methacrylic anhydride is 5 g: 1.5 mL. And / or, in step S1, the acylation reaction is carried out at a temperature of 0 °C for a time of 4 h; And / or, in step S1, in the first dialysis treatment, the dialysis bag used has a molecular weight cutoff of 3000 Da, deionized water is used as the dialysis solution, the dialysis time is 48 h, and the dialysis solution is changed every 8 h during the dialysis process; And / or, in step S1, the first freeze-drying process includes: pre-freezing at -20 ℃ for 24 h, and then freeze-drying at a vacuum of 10 Pa and a cold trap temperature of -20 ℃ for 24 h.

[0010] The specific steps of step S2 in the preparation method of the soluble microneedle patch for the treatment of bacterial keratitis as described above are as follows: Sodium selenite aqueous solution, chondroitin sulfate aqueous solution and L-ascorbic acid aqueous solution were prepared separately. The sodium selenite aqueous solution and the chondroitin sulfate aqueous solution were mixed and stirred to obtain a mixed system. The L-ascorbic acid aqueous solution was added to the mixed system, and a second stirring treatment was performed. After a second dialysis treatment and a second freeze-drying treatment, loose and porous chondroitin sulfate-selenium nanoparticles were obtained.

[0011] In the preparation method of the soluble microneedle patch for the treatment of bacterial keratitis as described above, the concentration of the sodium selenite aqueous solution is 0.02 mol / L, the concentration of the chondroitin sulfate aqueous solution is 4 mg / mL, and the concentration of the L-ascorbic acid aqueous solution is 0.2 mol / L. And / or, the volume ratio of the sodium selenite aqueous solution, the chondroitin sulfate aqueous solution, and the L-ascorbic acid aqueous solution is 1:1:1; And / or, the first stirring treatment is: stirring at room temperature and a speed of 250-350 rpm for 30-50 min; And / or, the second stirring treatment is: stirring at room temperature, in the dark, at a speed of 250-350 rpm for 4 hours; And / or, in the second dialysis treatment, the dialysis bag used has a molecular weight cutoff of 2000-4000 Da, deionized water is used as the dialysate, the dialysis time is 48 h, and the dialysate is changed every 8 h during the dialysis process; And / or, the second freeze-drying process includes a pre-freeze-drying process and a main freeze-drying process performed sequentially; the pre-freeze-drying process is: pre-freezing at -20 ℃ for 24 h, and the main freeze-drying process is: freeze-drying at a vacuum degree ≤10 Pa and a cold trap temperature ≤-50 ℃ for 24 to 48 h.

[0012] In the preparation method of the soluble microneedle patch for the treatment of bacterial keratitis as described above, in step S3, the mass ratio of the crosslinkable matrix to the chondroitin sulfate-selenium nanoparticles is (5-10):1, and the mass of the photoinitiator accounts for 2%-5% of the mass of the crosslinkable matrix; And / or, in step S3, the blending process is as follows: first, stirring at room temperature and 250-350 rpm for 30 min, and then sonicating at room temperature and 100W power for 10-20 min; the sonication adopts an intermittent sonication mode, wherein the intermittent sonication mode is paused for 2 s every 3 s of sonication.

[0013] In the preparation method of the soluble microneedle patch for the treatment of bacterial keratitis as described above, in step S4, the defoaming treatment is performed by defoaming for 20 to 30 minutes at room temperature and a vacuum degree ≤10 Pa. And / or, in step S4, the photocrosslinking curing is performed at a wavelength of 365 nm and a wavelength of 30 mW / cm². 2 Under light intensity conditions, perform ultraviolet crosslinking curing for 2–3 minutes; And / or, in step S4, the drying process is: drying at 37 °C for 12 h.

[0014] A third aspect of the present invention provides the use of a soluble microneedle patch in the preparation of a drug for treating bacterial keratitis, wherein the soluble microneedle patch is the aforementioned soluble microneedle patch for treating bacterial keratitis, or a soluble microneedle patch prepared by the aforementioned preparation method.

[0015] Compared with the prior art, the solution of the present invention has at least the following effects: 1. High-efficiency delivery advantage: The soluble microneedle patch for the treatment of bacterial keratitis provided by this invention is an integrated soluble microneedle. The microneedle structure can penetrate the corneal barrier in a minimally invasive manner, realizing a safe and efficient integrated treatment for bacterial keratitis. It breaks through the bottleneck of poor drug penetration and short residence time on the ocular surface of traditional eye drops, accurately delivering active ingredients to the site of corneal infection, improving the bioavailability of active ingredients, and reducing the frequency and dosage of drug administration.

[0016] 2. Multiple functions including antibacterial, anti-inflammatory, and promoting corneal tissue repair: This invention uses the product obtained by the acylation reaction of sodium chondroitin sulfate and methacrylic anhydride as a crosslinkable matrix. Chondroitin sulfate-selenium nanoparticles are highly dispersed in the three-dimensional crosslinked network formed by the crosslinkable matrix to prepare a soluble microneedle patch. This soluble microneedle patch has the advantages of excellent in vitro dissolution performance, excellent biocompatibility, excellent antibacterial activity, and excellent mechanical properties. It can achieve long-term sustained release of active ingredients, and its dissolution rate can be controlled by the solid content. It can effectively inhibit the growth of Staphylococcus aureus, has sufficient mechanical strength, and can penetrate the corneal barrier minimally invasively to achieve integrated, safe, and efficient treatment of bacterial keratitis, reducing systemic side effects and the risk of bacterial resistance.

[0017] 3. Simple preparation process: The present invention adopts solution blending and photocrosslinking curing technology. The process is simple, controllable, and repeatable. It does not require complex equipment and is suitable for large-scale production. Moreover, the mechanical properties of the microneedles after molding are adapted to the corneal puncture requirements (the single needle breaking force of the soluble microneedle patch in the embodiment of the present invention is >0.5 N). It is convenient to use and has broad prospects for clinical application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 This is a particle size-potential diagram of the chondroitin sulfate-selenium nanoparticles in Example 1 of the present invention; Figure 2 These are TEM images of the chondroitin sulfate-selenium nanoparticles obtained in Example 1 and the selenium nanoparticles obtained in Example 4 of this invention. Figure 2 Image a is a TEM image of chondroitin sulfate-selenium nanoparticles at a scale of 500 nm. Figure 2 b is a TEM image of chondroitin sulfate-selenium nanoparticles at a scale of 200 nm. Figure 2 c is a TEM image of selenium nanoparticles at a scale of 500 nm. Figure 2 d is a TEM image of selenium nanoparticles at a scale of 200 nm; Figure 3 This is an elemental mapping diagram of the chondroitin sulfate-selenium nanoparticles in Example 1 of the present invention, wherein, Figure 3 Image a is a TEM image of chondroitin sulfate-selenium nanoparticles at a scale bar of 200 nm. Figure 3Image b is a HAADF-STEM image of chondroitin sulfate-selenium nanoparticles. Figure 3 c represents the selenium (Se) mapping diagram. Figure 3 d represents the carbon (C) mapping diagram. Figure 3 The 'e' represents the oxygen (O) mapping diagram. Figure 3 f represents the HAADF-STEM image and surface distribution map of selenium, carbon, and oxygen elements; Figure 4 The figures show the 1H NMR and IR spectra of the crosslinkable matrix (ChS-MA) in Example 1 of this invention. Figure 4 α is the proton NMR spectrum of the crosslinkable matrix (ChS-MA). Figure 4 b is the infrared spectrum of chondroitin sulfate sodium (ChS) and crosslinkable matrix (ChS-MA); Figure 5 Image 'a' is a physical image of the soluble microneedle patch in Embodiment 1 of this invention. Figure 5 b is a microscopic morphology diagram of the needle tip of the soluble microneedle patch in Embodiment 1 of the present invention; Figure 6 These are the dissolution and release test results of the soluble microneedle patches in Examples 1-3 of this invention; Figure 7 The results of the biocompatibility test of the microneedle patch in Comparative Example 1 of this invention; Figure 8 The results of the biocompatibility test of the soluble microneedle patch in Example 2 of this invention; Figure 9 This is a diagram showing the inhibition zones of soluble microneedle patches at different concentrations in Example 2 of the present invention. Figure 10 This is an antibacterial coating diagram of soluble microneedle patches of different concentrations in Example 2 of the present invention; Figure 11 This is a diagram showing the mechanical properties of the soluble microneedle patch in Embodiment 2 of the present invention. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this art or in accordance with the product instructions. Reagents or instruments used without specified manufacturers are all conventional products that can be obtained commercially.

[0021] It should be noted that the use of terms such as "first" and "second" in this invention is for distinguishing similar objects and not for describing a specific order or sequence, and therefore should not be construed as a limitation of this invention.

[0022] In the following description, the term "and / or" is used to describe the relationship between related objects, indicating that there can be three relationships. For example, A and / or B can mean: A exists alone, B exists alone, and A and B exist simultaneously. A and B can be singular or plural.

[0023] Those skilled in the art should understand that, in the following description of the embodiments of the present invention, the sequence of numbers does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present invention.

[0024] Those skilled in the art will understand that the numerical ranges in the embodiments of the present invention should be understood to specifically disclose each intermediate value between the upper and lower limits of the range. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, the technical / scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] A first aspect of the present invention provides a soluble microneedle patch for the treatment of bacterial keratitis, the soluble microneedle patch comprising: A crosslinkable matrix, wherein the crosslinkable matrix is ​​photocrosslinked and cured to form a three-dimensional crosslinked network; Chondroitin sulfate-selenium nanoparticles, wherein the chondroitin sulfate-selenium nanoparticles form a dispersed phase in a three-dimensional cross-linked network formed by the cross-linkable matrix; The structural formula of the crosslinkable matrix is ​​as follows: .

[0027] This invention uses the product obtained by acylation reaction of sodium chondroitin sulfate and methacrylic anhydride as a crosslinkable matrix, and chondroitin sulfate-selenium nanoparticles as the dispersed phase. The chondroitin sulfate-selenium nanoparticles are dispersed in a three-dimensional crosslinked network formed by the crosslinkable matrix to prepare a soluble microneedle patch. This soluble microneedle patch has the advantages of excellent in vitro dissolution performance, excellent biocompatibility, excellent antibacterial activity, and excellent mechanical properties. It can achieve long-term sustained release of active ingredients, and its dissolution rate can be controlled by the solid content. It can effectively inhibit the growth of Staphylococcus aureus, has sufficient mechanical strength, and can penetrate the corneal barrier minimally invasively, realizing a safe and efficient integrated treatment for bacterial keratitis.

[0028] In some embodiments, the mass ratio of the crosslinkable matrix to the chondroitin sulfate-selenium nanoparticles is (5-10):1.

[0029] When the mass ratio of the crosslinkable matrix to the chondroitin sulfate-selenium nanoparticles meets the above conditions, an integrated soluble microneedle patch with excellent in vitro solubility, excellent biocompatibility, excellent antibacterial activity, and excellent mechanical properties can be successfully prepared.

[0030] In some embodiments, the chondroitin sulfate-selenium nanoparticles have an average particle size of 120–130 nm, a polydispersity index (PDI) of 0.05–0.1, and a zeta potential of -41–-38 mV.

[0031] When the average particle size, polydispersity index (PDI), and zeta potential of the chondroitin sulfate-selenium nanoparticles meet the above conditions, the dispersion stability of the chondroitin sulfate-selenium nanoparticles is improved, preventing their aggregation and sedimentation. This facilitates their uniform dispersion in the three-dimensional cross-linked network formed by the cross-linkable matrix, resulting in an integrated soluble microneedle patch with excellent in vitro solubility, excellent biocompatibility, excellent antibacterial activity, and excellent mechanical properties.

[0032] In some embodiments, the soluble microneedle patch is an integral soluble microneedle, comprising a substrate and needle tips arranged in an array on the substrate.

[0033] In this invention, the soluble microneedle patch is an integrated soluble microneedle, and the substrate and the needle tip are made of the same material, which fundamentally eliminates problems such as delamination and weak bonding that may occur between different materials, avoids possible chemical reactions or interactions between different materials, and significantly improves biocompatibility and safety.

[0034] The present invention does not impose a particular limitation on the specific shape of the substrate, which can be selected according to actual needs. For example, the shape of the substrate can be circular, square, etc. The present invention also does not impose a particular limitation on the specific dimensions of the substrate, which can be selected according to actual needs. Further, when the substrate is circular, its diameter is 5–10 mm and its thickness is 1–3 mm.

[0035] In this invention, the needle tips are arranged in an array on the substrate. The specific shape of the array is not particularly limited and can be selected according to actual needs; for example, the array can be a circular array, a linear array, etc.

[0036] In some embodiments, the needle tip is conical in shape, with a needle height of 590–610 μm and a needle base diameter of 290–310 μm.

[0037] The present invention does not impose a specific limit on the number of needle tips, which can be selected according to actual needs.

[0038] A second aspect of the present invention provides a method for preparing the soluble microneedle patch for the treatment of bacterial keratitis, comprising the following steps: S1. Chondroitin sulfate sodium is dissolved in water and then methacrylic anhydride is added to carry out an acylation reaction. The product after the acylation reaction is subjected to a first dialysis treatment and a first freeze-drying treatment to obtain a loose and porous crosslinkable matrix. S2. Using sodium selenite as the selenium source, chondroitin sulfate as the stabilizer and capping agent, and L-ascorbic acid as the reducing agent, chondroitin sulfate-selenium nanoparticles are generated in situ through coordination, electrostatic adsorption and steric hindrance effect. S3. The crosslinkable matrix is ​​added to water to form a uniform and transparent crosslinkable matrix aqueous solution. The photoinitiator and the chondroitin sulfate-selenium nanoparticles are added to the crosslinkable matrix aqueous solution in sequence and then blended to obtain a microneedle precursor solution. S4. The microneedle precursor liquid is injected into the microneedle mold, and after degassing, photocrosslinking curing, and drying, the soluble microneedle patch is obtained.

[0039] The present invention can produce soluble microneedle patches with excellent in vitro solubility, excellent biocompatibility, excellent antibacterial activity and excellent mechanical properties through the above preparation method.

[0040] In step S1, the role of the crosslinkable matrix is ​​as follows: the molecular chains of the crosslinkable matrix can form a large number of intermolecular hydrogen bonds through hydroxyl, carboxyl, and amide groups, and are supplemented by electrostatic interactions. Multiple molecular chains are effectively connected and entangled, and synergistically construct a three-dimensional crosslinked network. This three-dimensional crosslinked network provides a stable dispersion environment for the dispersed phase (chondroitin sulfate-selenium nanoparticles) and prevents its agglomeration and sedimentation. On the other hand, it lays a good foundation for the overall formability and mechanical properties of the material, and provides necessary support for the successful preparation of soluble microneedle patches.

[0041] The technical principle used in step S2 of this invention to prepare chondroitin sulfate-selenium nanoparticles is explained as follows: Chondroitin sulfate guides the selenium element in the selenium source to form selenium nanocrystals in situ through coordination, and the surface of the selenium nanocrystals is coated by electrostatic adsorption. Then, the long chain structure provides a steric hindrance effect. The three work together to finally form chondroitin sulfate-selenium nanoparticles with uniform particle size and stable dispersion.

[0042] In step S3, the blending treatment is to ensure that the components are evenly dispersed, so that the chondroitin sulfate-selenium nanoparticles can be evenly dispersed in the three-dimensional cross-linked network formed by the cross-linkable matrix after subsequent photocrosslinking and curing.

[0043] In step S4, the degassing treatment serves to remove air bubbles from the microneedle precursor solution, ensuring the formation of a soluble microneedle patch with a complete morphology; the photocrosslinking and curing treatment serves to enable the crosslinkable matrix to undergo photopolymerization to form a stable three-dimensional crosslinked network, and to disperse chondroitin sulfate-selenium nanoparticles in the three-dimensional crosslinked network; the drying treatment is to remove residual moisture from the soluble microneedle patch.

[0044] In some embodiments, in step S1, the ratio of sodium chondroitin sulfate to methacrylic anhydride is 5 g: 1.5 mL.

[0045] In some embodiments, in step S1, the acylation reaction is carried out at a temperature of 0 °C for a time of 4 h.

[0046] In some embodiments, in step S1, the dialysis bag used in the first dialysis treatment has a molecular weight cutoff of 3000 Da, deionized water is used as the dialysis solution, the dialysis time is 48 h, and the dialysis solution is replaced every 8 h during the dialysis process.

[0047] In some embodiments, step S1, the first freeze-drying process includes: pre-freezing at -20 ℃ for 24 h, and then freeze-drying at a vacuum of 10 Pa and a cold trap temperature of -20 ℃ for 24 h.

[0048] In some implementations, step S2 specifically involves the following steps: Sodium selenite aqueous solution, chondroitin sulfate aqueous solution and L-ascorbic acid aqueous solution were prepared separately. The sodium selenite aqueous solution and the chondroitin sulfate aqueous solution were mixed and stirred to obtain a mixed system. The L-ascorbic acid aqueous solution was added to the mixed system, and a second stirring treatment was performed. After a second dialysis treatment and a second freeze-drying treatment, loose and porous chondroitin sulfate-selenium nanoparticles were obtained.

[0049] In some embodiments, the concentration of the sodium selenite aqueous solution is 0.02 mol / L, the concentration of the chondroitin sulfate aqueous solution is 4 mg / mL, and the concentration of the L-ascorbic acid aqueous solution is 0.2 mol / L.

[0050] In some embodiments, the volume ratio of the sodium selenite aqueous solution, the chondroitin sulfate aqueous solution, and the L-ascorbic acid aqueous solution is 1:1:1.

[0051] In some embodiments, the first stirring process is: stirring at room temperature and a speed of 250-350 rpm for 30-50 minutes.

[0052] In some embodiments, the second stirring treatment is: stirring at room temperature, in the dark, at a speed of 250-350 rpm for 4 hours.

[0053] In some embodiments, in the second dialysis treatment, the dialysis bag used has a molecular weight cutoff of 2000-4000 Da, deionized water is used as the dialysate, the dialysis time is 48 hours, and the dialysate is changed every 8 hours during the dialysis process. The dialysis treatment is to remove unreacted small molecule impurities, salts, and L-ascorbic acid.

[0054] In some embodiments, the second freeze-drying process includes a pre-freeze-drying process and a main freeze-drying process performed sequentially; the pre-freeze-drying process is: pre-freezing at -20 ℃ for 24 h, and the main freeze-drying process is: freeze-drying at a vacuum degree ≤10 Pa and a cold trap temperature ≤-50 ℃ for 24 to 36 h.

[0055] In some embodiments, in step S3, the mass ratio of the crosslinkable matrix to the chondroitin sulfate-selenium nanoparticles is (5-10):1, and the mass of the photoinitiator accounts for 2%-5% of the mass of the crosslinkable matrix.

[0056] In some embodiments, in step S3, the blending process is as follows: first, stirring at room temperature and 250-350 rpm for 30 min, and then sonicating at room temperature and 100W power for 10-20 min; the sonication adopts an intermittent sonication mode, wherein the intermittent sonication mode is paused for 2 s every 3 s of sonication.

[0057] In this invention, the components are first stirred at room temperature and 250–350 rpm for 30 min to ensure thorough mixing, dissolution, and pre-dispersion. Then, the components are sonicated at room temperature and 100W power for 10–20 min to refine the selenium nanocrystal nuclei in the chondroitin sulfate-selenium nanoparticles using the ultrasonic cavitation effect. This process also enhances the coordination, coating, and stabilization of the selenium nanocrystal nuclei by chondroitin sulfate, facilitating the uniform dispersion of the chondroitin sulfate-selenium nanoparticles in the three-dimensional cross-linked network formed by the cross-linkable matrix after subsequent processing.

[0058] In some embodiments, in step S4, the degassing treatment is performed by degassing for 20 to 30 minutes at room temperature and a vacuum degree ≤10 Pa.

[0059] When the degassing treatment meets the above conditions, it effectively removes air bubbles from the microneedle precursor solution, ensuring that the microneedle precursor solution completely fills the microneedle mold and guarantees the formation of a morphologically intact soluble microneedle patch. Without degassing, air bubbles in the microneedle precursor solution, after photocrosslinking and curing in the microneedle mold, will form voids or defects. This leads to incomplete needle tips and flawed microneedles, making it impossible to obtain a morphologically intact soluble microneedle patch. The integrity of the morphology of the soluble microneedle patch is fundamental to its effectiveness. Air bubbles are stress concentration points within the material, significantly weakening the mechanical strength of the soluble microneedle patch. When using soluble microneedle patches with voids or defects, they are prone to bending or even breakage, failing to guarantee sufficient puncture strength. Furthermore, the drug or active ingredient content varies between each microneedle and even between different areas of the same microneedle, resulting in unstable drug or active ingredient release and failing to achieve the expected therapeutic effect.

[0060] In some embodiments, in step S4, the photocrosslinking curing is performed at a wavelength of 365 nm and a wavelength of 30 mW / cm². 2 Under light intensity conditions, perform ultraviolet cross-linking curing for 2–3 minutes.

[0061] When photocrosslinking curing meets the above conditions, the crosslinkable matrix undergoes a photopolymerization reaction to form a stable three-dimensional crosslinked network, and chondroitin sulfate-selenium nanoparticles are dispersed in the three-dimensional crosslinked network.

[0062] In some embodiments, in step S4, the drying process is: drying at 37 °C for 12 h.

[0063] When the drying process meets the above conditions, removing residual moisture without damaging the soluble microneedle patch can greatly inhibit reactions such as oxidation, prevent the deactivation of its active ingredients, and further improve the stability of the soluble microneedle patch.

[0064] A third aspect of the present invention provides the use of a soluble microneedle patch in the preparation of a drug for treating bacterial keratitis, wherein the soluble microneedle patch is the aforementioned soluble microneedle patch for treating bacterial keratitis, or a soluble microneedle patch prepared by the aforementioned preparation method.

[0065] The present invention will be further described below through specific embodiments.

[0066] Example 1

[0067] The soluble microneedle patch provided in this embodiment is prepared by a method including the following steps: (1) Preparation of crosslinkable matrix: Dissolve 5 g of sodium chondroitin sulfate in 100 mL of water, add 1.5 mL of methacrylic anhydride, and carry out an acylation reaction at 0 °C for 4 h to obtain the reaction product. The reaction product was placed in a dialysis bag with a molecular weight cutoff of 3000 Da, and deionized water was used as the dialysate. The dialysate was dialyzed at room temperature for 48 h, and the dialysate was changed every 8 h during the process to fully remove unreacted small molecules and impurities, and the purified product was obtained. The purified product was pre-frozen in an ultra-low temperature freezer at -20 ℃ for 24 h, and then transferred to a vacuum freeze dryer and freeze-dried at a vacuum degree of 10 Pa and a cold trap temperature of -20 ℃ for 24 h to completely remove moisture and obtain a loose and porous crosslinkable matrix.

[0068] (2) In-situ generation of chondroitin sulfate-selenium nanoparticles: Weigh 0.197 g of sodium selenite, add 50 mL of deionized water, and stir at room temperature and 300 rpm for 15 min until completely dissolved to obtain a sodium selenite aqueous solution with a concentration of 0.02 mol / L (denoted as solution A). Weigh 0.2 g of sodium chondroitin sulfate, add 50 mL of deionized water, and stir at room temperature and 250 rpm for 30 min until the system is homogeneous and transparent to obtain an aqueous solution of chondroitin sulfate with a concentration of 4 mg / mL (referred to as solution B). Dissolve 0.352 g of L-ascorbic acid in 50 mL of deionized water to prepare an L-ascorbic acid aqueous solution with a concentration of 0.2 mol / L (denoted as solution C). Add 50 mL of solution A and 50 mL of solution B to a 1000 mL single-necked flask, place it on a magnetic stirrer, and stir for 40 min at room temperature and 300 rpm to achieve pre-complexation of chondroitin sulfate and selenite to obtain a mixed system. Under continuous stirring and light-protected conditions, 50 mL of solution C was added dropwise to the mixture at a rate of 1 mL / min using a constant pressure dropping funnel. After the addition was complete, the mixture was stirred for 4 h at room temperature, in the dark, and at a speed of 300 rpm. The system gradually changed from colorless to a uniform orange-red color, yielding the crude product of chondroitin sulfate-selenium nanoparticles. The crude chondroitin sulfate-selenium nanoparticles were transferred into a dialysis bag with a molecular weight cutoff of 3500 Da. Deionized water was used as the dialysis solution, and the dialysis time was 48 hours. During the dialysis process, the deionized water was replaced every 8 hours to remove unreacted small molecule impurities, salts, and excess L-ascorbic acid, thus obtaining the product. The product was transferred into centrifuge tubes and pre-frozen in an ultra-low temperature freezer at -20 ℃ for 24 h. Then it was transferred to a vacuum freeze dryer and freeze-dried for 48 h under a vacuum of 10 Pa and a cold trap temperature of -50 ℃ to form a loose and porous orange-red solid powder, which is chondroitin sulfate-selenium nanoparticles.

[0069] (3) Preparation of microneedle precursor solution: Add 0.5 g of the crosslinkable matrix from step (1) to 10 mL of deionized water and stir for 60 min at room temperature and 300 rpm to form a homogeneous and transparent crosslinkable matrix aqueous solution. 0.025 g of photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphine) was added to the crosslinkable matrix aqueous solution and stirred continuously for 30 min until completely dissolved to obtain a mixture. Add 0.1 g of the chondroitin sulfate-selenium nanoparticles from step (2) to the mixture, stir for 30 min at room temperature and 300 rpm, and then sonicate for 15 min at room temperature and 100 W power to uniformly disperse the chondroitin sulfate-selenium nanoparticles and obtain the microneedle precursor solution; wherein, the sonication adopts an intermittent sonication mode, and the intermittent sonication mode is paused for 2 s every 3 s of sonication.

[0070] (4) Preparation of soluble microneedle patches: The microneedle precursor solution was injected into the PDMS microneedle mold and degassed for 30 minutes at room temperature and a vacuum of 10 Pa to remove air bubbles from the microneedle precursor solution, ensuring that the microneedle precursor solution completely filled the PDMS microneedle mold. Excess precursor solution was wiped off the surface of the PDMS microneedle mold with lint-free paper to obtain a PDMS microneedle mold filled with microneedle precursor solution. The PDMS microneedle mold filled with the microneedle precursor solution was placed under a UV crosslinker at a wavelength of 365 nm and a wavelength of 30 mW / cm². 2 Under light intensity conditions, UV crosslinking and curing were performed for 2 min to induce photopolymerization of the crosslinkable matrix, forming a stable three-dimensional crosslinked network. After crosslinking, the product was dried in a vacuum drying oven at 37 ℃ for 12 h. The molded product was then removed from the PDMS microneedle mold to obtain a soluble microneedle patch with a solid content of 5%. The soluble microneedle patch is an integral soluble microneedle, comprising a substrate and needle tips arranged in a ring array on the substrate. The substrate is circular with a diameter of 8 mm and a thickness of 1.5 mm. The needle tips are conical with a height of 600 μm and a base diameter of 300 μm. The number of needle tips is 70.

[0071] Example 2

[0072] The preparation method of the soluble microneedle patch provided in this embodiment is basically the same as that in embodiment 1, except that in step (3): in step (3), "0.5 g of the crosslinkable matrix in step (1) is added to 10 mL of deionized water" is replaced with "0.8 g of the crosslinkable matrix in step (1) is added to 10 mL of deionized water".

[0073] The preparation method of this embodiment finally yielded a soluble microneedle patch with a solid content of 8%.

[0074] Example 3

[0075] The preparation method of the soluble microneedle patch provided in this embodiment is basically the same as that in embodiment 1, except that in step (3): in step (3), "0.5 g of the crosslinkable matrix in step (1) is added to 10 mL of deionized water" is replaced with "1 g of the crosslinkable matrix in step (1) is added to 10 mL of deionized water".

[0076] The preparation method of this embodiment finally yielded a soluble microneedle patch with a solid content of 10%.

[0077] Example 4

[0078] This embodiment provides a selenium nanoparticle, the preparation method of which includes: Weigh 0.197 g of sodium selenite, add 50 mL of deionized water, and stir at room temperature and 300 rpm for 15 min until completely dissolved to obtain a sodium selenite aqueous solution with a concentration of 0.02 mol / L (denoted as solution A). Dissolve 0.352 g of L-ascorbic acid in 50 mL of deionized water to prepare an L-ascorbic acid aqueous solution with a concentration of 0.2 mol / L (referred to as solution B). Under continuous stirring and light-protected conditions, 50 mL of solution B was added dropwise to solution A at a rate of 1 mL / min using a constant pressure dropping funnel. After the addition was completed, the mixture was stirred for 4 h at room temperature, in the dark, and at a speed of 300 rpm. The system gradually changed from colorless to a uniform orange-red color, yielding the crude selenium nanoparticle product. The crude selenium nanoparticle product was transferred into a dialysis bag with a molecular weight cutoff of 3500 Da and immersed in 5L of deionized water for dialysis treatment. The dialysis time was 48h, and the deionized water was replaced every 8h during the dialysis process to remove unreacted small molecule impurities, salts and excess L-ascorbic acid to obtain the product. The product was transferred into centrifuge tubes and pre-frozen in an ultra-low temperature freezer at -20 °C for 24 h. Then it was transferred to a vacuum freeze dryer and freeze-dried for 48 h under a vacuum of 10 Pa and a cold trap temperature of -50 °C to obtain selenium nanoparticles.

[0079] Comparative Example 1 (Chondroitin sulfate-selenium nanoparticles without addition) The microneedle patch provided in this comparative example was prepared by a method including the following steps: (1) Preparation of crosslinkable matrix: Same as step (1) in Example 1.

[0080] (2) Preparation of microneedle precursor solution: Add 0.8 g of the crosslinkable matrix from step (1) to 10 mL of deionized water and stir for 60 min at room temperature and 300 rpm to form a homogeneous and transparent crosslinkable matrix aqueous solution. 0.025 g of photoinitiator LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphine) was added to the crosslinkable matrix aqueous solution and stirred continuously for 30 min until completely dissolved to obtain microneedle precursor solution.

[0081] (4) Preparation of microneedle patches: The microneedle precursor solution was injected into the PDMS microneedle mold and degassed for 30 minutes at room temperature and a vacuum of 10 Pa to remove air bubbles from the microneedle precursor solution, ensuring that the microneedle precursor solution completely filled the PDMS microneedle mold. Excess precursor solution was wiped off the surface of the PDMS microneedle mold with lint-free paper to obtain a PDMS microneedle mold filled with microneedle precursor solution. The PDMS microneedle mold filled with the microneedle precursor solution was placed under a UV crosslinker at a wavelength of 365 nm and a wavelength of 30 mW / cm². 2Under light intensity conditions, UV crosslinking and curing were performed for 2 min to allow the crosslinkable matrix to undergo photopolymerization and form a stable three-dimensional crosslinked network. After crosslinking, the product was dried in a vacuum drying oven at 37 ℃ for 12 h. The molded product was then removed from the PDMS microneedle mold to obtain a microneedle patch. The microneedle patch includes a substrate and needle tips arranged in a ring array on the substrate. The substrate is circular with a diameter of 8 mm and a thickness of 1.5 mm. The needle tips are conical with a height of 600 μm and a base diameter of 300 μm. The number of needle tips is 70.

[0082] Performance testing 1. The chondroitin sulfate-selenium nanoparticles obtained in Example 1 of this invention were tested for particle size and zeta potential. The results are as follows: Figure 1 As shown. Figure 1 This is a particle size-potential diagram of the chondroitin sulfate-selenium nanoparticles in Example 1 of the present invention.

[0083] Depend on Figure 1 It is known that the average particle size of chondroitin sulfate-selenium nanoparticles is 120.93 nm, the polydispersity index (PDI) is 0.133, and the zeta potential is -35.23 mV.

[0084] 2. The chondroitin sulfate-selenium nanoparticles obtained in Example 1 and the selenium nanoparticles obtained in Example 4 of this invention were tested by transmission electron microscopy (TEM), and the results are as follows: Figure 2 As shown. Figure 2 These are TEM images of the chondroitin sulfate-selenium nanoparticles obtained in Example 1 and the selenium nanoparticles obtained in Example 4 of this invention. Figure 2 Image a is a TEM image of chondroitin sulfate-selenium nanoparticles at a scale of 500 nm. Figure 2 b is a TEM image of chondroitin sulfate-selenium nanoparticles at a scale of 200 nm. Figure 2 c is a TEM image of selenium nanoparticles at a scale of 500 nm. Figure 2 d is a TEM image of selenium nanoparticles at a scale of 200 nm.

[0085] Depend on Figure 2 As can be seen from ab, chondroitin sulfate-selenium nanoparticles have a highly monodisperse state and do not form clusters. The particles have a uniform morphology and clear outline.

[0086] Depend on Figure 2 As can be seen from the CD, the selenium nanoparticles form clusters with uneven morphology.

[0087] 3. Elemental mapping tests were performed on the chondroitin sulfate-selenium nanoparticles obtained in Example 1 of this invention, and the results are as follows: Figure 3As shown. Figure 3 This is an elemental mapping diagram of the chondroitin sulfate-selenium nanoparticles in Example 1 of the present invention, wherein, Figure 3 Image a is a TEM image of chondroitin sulfate-selenium nanoparticles at a scale bar of 200 nm. Figure 3 Image b is a HAADF-STEM image of chondroitin sulfate-selenium nanoparticles. Figure 3 c represents the selenium (Se) mapping diagram. Figure 3 d represents the carbon (C) mapping diagram. Figure 3 The 'e' represents the oxygen (O) mapping diagram. Figure 3 f represents the HAADF-STEM image and the surface distribution map of selenium, carbon, and oxygen elements.

[0088] Depend on Figure 3 It can be seen that the signals of the three elements Se, C and O are evenly distributed and their positions are highly overlapping, indicating that the prepared chondroitin sulfate-selenium nanoparticles have good dispersibility.

[0089] 4. The crosslinkable matrix obtained in Example 1 of this invention was subjected to NMR and IR spectroscopy tests, and the results are as follows: Figure 4 As shown. Figure 4 The figures show the 1H NMR and IR spectra of the crosslinkable matrix (ChS-MA) in Example 1 of this invention. Figure 4 α is the proton NMR spectrum of the crosslinkable matrix (ChS-MA). Figure 4 b is the infrared spectrum of chondroitin sulfate sodium (ChS) and crosslinkable matrix (ChS-MA).

[0090] Depend on Figure 4 As can be seen from a, the proton spectrum of the crosslinkable matrix (ChS-MA) is... 1 The H NMR results showed characteristic proton peaks of the methacryloyl group at 5.5-6.5 ppm and 1.8-2.2 ppm, indicating that the methacryloyl group has been successfully grafted onto the chondroitin sulfate molecular chain, and the crosslinkable matrix can undergo crosslinking to form a gel.

[0091] Depend on Figure 4 As can be seen from b, the crosslinkable matrix (ChS-MA) has a crosslinkable structure.

[0092] The above results demonstrate the successful preparation of a crosslinkable matrix with the following structural formula: .

[0093] 5. The morphology of the soluble microneedle patch obtained in Example 1 of this invention was tested, and the results are as follows: Figure 5 As shown. Figure 5Image 'a' is a physical image of the soluble microneedle patch in Embodiment 1 of this invention. Figure 5 b is a microscopic morphology diagram of the needle tip of the soluble microneedle patch in Embodiment 1 of the present invention.

[0094] Depend on Figure 5 As can be seen from a, the soluble microneedle patch is an integrated soluble microneedle, consisting of a substrate and needle tips arranged in a ring array on the substrate. The needle tips are neatly arranged, have a uniform appearance, and are free from collapse or damage, which proves that the molding is good.

[0095] Depend on Figure 5 As can be seen from b, the soluble microneedle patch has a sharp tip, a straight needle body, and a smooth surface, proving its excellent structure.

[0096] 6. Dissolution and release tests were performed on the soluble microneedle patches obtained in Examples 1-3 of this invention: (1) In vitro dissolution experiment: Soluble microneedle patch samples were placed in 24-well plates, and PBS solution or physiological saline (0.9% NaCl) was added to completely submerge the soluble microneedle patches. The plates were then incubated at a constant temperature of 37 °C, and the dissolution of the soluble microneedle patches was observed and recorded at preset time points.

[0097] (2) Agar-simulated skin dissolution experiment: A 2% solid content agarose gel was used as the skin simulation matrix and placed in a 24-well plate. Soluble microneedle patch samples were vertically pressed into the agarose gel and incubated at 37°C. The patches were then removed at preset time points, and the dissolution of the soluble microneedle patches was observed and recorded.

[0098] The soluble microneedle patches from Examples 1-3 of this invention were used as samples for in vitro dissolution experiments and agar-simulated skin dissolution experiments to evaluate the dissolution rate of soluble microneedle patches with different solid contents. The results are as follows: Figure 6 As shown. Figure 6 These are the dissolution and release test results of the soluble microneedle patches in Examples 1-3 of this invention.

[0099] Depend on Figure 6 It is known that the soluble microneedle patch provided in the embodiments of the present invention can be dissolved and released in PBS, physiological saline and agar. Soluble microneedle patches with different solid contents have different dissolution rates, indicating that the soluble microneedle patch provided in the embodiments of the present invention has excellent in vitro dissolution performance, and the dissolution rate can be controlled by the solid content, which meets the application requirements of soluble microneedles.

[0100] 7. Biocompatibility tests were performed on the soluble microneedle patch in Example 2 of the present invention and the microneedle patch in Comparative Example 1, respectively: The microneedle patch from Comparative Example 1 was mixed with pure water to prepare microneedle matrices of different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL).

[0101] The soluble microneedle patch from Example 2 of this invention was mixed with pure water to prepare ChS-Se NPs at different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL).

[0102] Experimental methods: Mouse fibroblasts (L929) were seeded into 96-well plates and incubated at 37 ℃ in a 5% CO2 incubator for 24 h to allow cell adhesion. Then, microneedle patch samples were added as the experimental group, and those without microneedle patch samples served as the control. After culturing for another 24 h, CCK-8 solution was added to each well, and the cells were incubated in the dark for 2 h. The absorbance (OD value) at 450 nm was measured using a microplate reader, and the relative cell viability was calculated.

[0103] Different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL) of microneedle matrix and different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL) of ChS-Se NPs were used as microneedle patch samples, and the biocompatibility of the microneedle patches was evaluated according to the above experimental method. The results are as follows: Figures 7-8 As shown. Figure 7 The results of the biocompatibility test of the microneedle patch in Comparative Example 1 of this invention; Figure 8 The results are the biocompatibility test results of the soluble microneedle patch in Example 2 of this invention.

[0104] Depend on Figures 7-8 It can be seen that, compared with the microneedle patch in Comparative Example 1, the soluble microneedle patch in Example 2 of the present invention has a higher cell survival rate, lower cytotoxicity, and better biocompatibility, meeting the basic requirements for biomedical applications.

[0105] 8. The antibacterial activity of the soluble microneedle patch obtained in Example 2 of this invention was tested: (1) Inhibition zone experiment: The soluble microneedle patch from Example 2 of this invention was mixed with pure water to prepare ChS-Se NPs at different concentrations (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL).

[0106] Experimental Methods: Staphylococcus aureus culture in the logarithmic growth phase was evenly spread onto LB agar plates. Different concentrations (0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL) of ChS-Se NPs were then gently placed into the plates. The plates were incubated at 37 ℃ for 24 h. The diameter of the inhibition zone was observed and measured to evaluate the antibacterial activity. Results are as follows: Figure 9 . Figure 9 This is a diagram showing the inhibition zones of soluble microneedle patches at different concentrations in Example 2 of the present invention.

[0107] Depend on Figure 9 It is known that the soluble microneedle patch provided in the embodiments of the present invention has excellent antibacterial activity, can effectively inhibit the growth of Staphylococcus aureus, and the antibacterial effect can be controlled by concentration, showing good application potential in the treatment of bacterial keratitis.

[0108] (2) Antibacterial coating plate test: The soluble microneedle patch from Example 2 of this invention was mixed with pure water to prepare ChS-Se NPs at different concentrations (0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, 2 mg / mL).

[0109] Experimental Methods: Staphylococcus aureus culture in the logarithmic growth phase and microneedle patch samples were added to LB liquid medium as the experimental group. A blank group (without Staphylococcus aureus culture and microneedle patch samples) and a control group (with Staphylococcus aureus culture but without microneedle patch samples) were used. The mixture was thoroughly mixed and incubated at 37 ℃ for 12 h to obtain the cultured bacterial solution. The cultured bacterial solution was then evenly spread onto LB agar plates using a disposable spreader and incubated at 37 ℃ for 24 h. The colony distribution of Staphylococcus aureus was observed.

[0110] Different concentrations (0.25 mg / mL, 0.5 mg / mL, 1 mg / mL, 1.5 mg / mL, and 2 mg / mL) of ChS-SeNPs were used as microneedle patch samples. Antibacterial plating experiments were conducted according to the above experimental method to evaluate antibacterial activity. The results are as follows: Figure 10 As shown. Figure 10 This is an antibacterial coating diagram of soluble microneedle patches of different concentrations in Example 2 of the present invention.

[0111] Depend on Figure 10It can be seen that, compared with the control group, the number of Staphylococcus aureus colonies was significantly reduced after treatment with different concentrations of ChS-Se NPs, and the antibacterial effect was concentration-dependent, further indicating that the soluble microneedle patch provided in the embodiments of the present invention has excellent antibacterial activity and can effectively inhibit the growth of Staphylococcus aureus.

[0112] 9. The mechanical properties of the soluble microneedle patch obtained in Example 2 of this invention were tested, and the results are as follows: Figure 11 As shown. Figure 11 This is a mechanical property diagram of the soluble microneedle patch in Embodiment 2 of the present invention. Figure 11 It can be seen that the single needle breaking force of the soluble microneedle patch in Example 2 is >0.5 N, and the total strength of the 72-needle array is greater than 36 N, which shows excellent mechanical properties. This indicates that the soluble microneedle patch provided by the present invention has sufficient mechanical strength, can penetrate the corneal barrier in a minimally invasive manner, can achieve safe puncture, is not easy to break, has a stable structure, and meets the practical application requirements of minimally invasive ocular drug delivery.

[0113] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A soluble microneedle patch for the treatment of bacterial keratitis, characterized in that, The soluble microneedle patch includes: A crosslinkable matrix, wherein the crosslinkable matrix is ​​photocrosslinked and cured to form a three-dimensional crosslinked network; Chondroitin sulfate-selenium nanoparticles, wherein the chondroitin sulfate-selenium nanoparticles form a dispersed phase in a three-dimensional cross-linked network formed by the cross-linkable matrix; The structural formula of the crosslinkable matrix is ​​as follows: 。 2. The soluble microneedle patch for treating bacterial keratitis according to claim 1, characterized in that, The mass ratio of the crosslinkable matrix to the chondroitin sulfate-selenium nanoparticles is (5-10):1; And / or, the chondroitin sulfate-selenium nanoparticles have an average particle size of 120–130 nm, a polydispersity index (PDI) of 0.05–0.1, and a zeta potential of -41–-38 mV.

3. The soluble microneedle patch for treating bacterial keratitis according to claim 1, characterized in that, The soluble microneedle patch is an integrated soluble microneedle, comprising a substrate and needle tips arranged in an array on the substrate; The needle tip is conical in shape, with a needle height of 590–610 μm and a needle base diameter of 290–310 μm.

4. A method for preparing a soluble microneedle patch for the treatment of bacterial keratitis as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1. Chondroitin sulfate sodium is dissolved in water and then methacrylic anhydride is added to carry out an acylation reaction. The product after the acylation reaction is subjected to a first dialysis treatment and a first freeze-drying treatment to obtain a loose and porous crosslinkable matrix. S2. Using sodium selenite as the selenium source, chondroitin sulfate as the stabilizer and capping agent, and L-ascorbic acid as the reducing agent, chondroitin sulfate-selenium nanoparticles are generated in situ through coordination, electrostatic adsorption and steric hindrance effect. S3. The crosslinkable matrix is ​​added to water to form a uniform and transparent crosslinkable matrix aqueous solution. The photoinitiator and the chondroitin sulfate-selenium nanoparticles are added to the crosslinkable matrix aqueous solution in sequence and then blended to obtain a microneedle precursor solution. S4. The microneedle precursor liquid is injected into the microneedle mold, and after degassing, photocrosslinking curing, and drying, the soluble microneedle patch is obtained.

5. The method for preparing the soluble microneedle patch for the treatment of bacterial keratitis according to claim 4, characterized in that, In step S1, the ratio of sodium chondroitin sulfate to methacrylic anhydride is 5 g: 1.5 mL; And / or, in step S1, the acylation reaction is carried out at a temperature of 0 °C for a time of 4 h; And / or, in step S1, in the first dialysis treatment, the dialysis bag used has a molecular weight cutoff of 3000 Da, deionized water is used as the dialysis solution, the dialysis time is 48 h, and the dialysis solution is changed every 8 h during the dialysis process; And / or, in step S1, the first freeze-drying process includes: pre-freezing at -20 ℃ for 24 h, and then freeze-drying at a vacuum of 10 Pa and a cold trap temperature of -20 ℃ for 24 h.

6. The method for preparing the soluble microneedle patch for the treatment of bacterial keratitis according to claim 4, characterized in that, The specific steps of step S2 are as follows: Sodium selenite aqueous solution, chondroitin sulfate aqueous solution and L-ascorbic acid aqueous solution were prepared separately. The sodium selenite aqueous solution and the chondroitin sulfate aqueous solution were mixed and stirred to obtain a mixed system. The L-ascorbic acid aqueous solution was added to the mixed system, and a second stirring treatment was performed. After a second dialysis treatment and a second freeze-drying treatment, loose and porous chondroitin sulfate-selenium nanoparticles were obtained.

7. The method for preparing the soluble microneedle patch for the treatment of bacterial keratitis according to claim 6, characterized in that, The concentration of the sodium selenite aqueous solution is 0.02 mol / L, the concentration of the chondroitin sulfate aqueous solution is 4 mg / mL, and the concentration of the L-ascorbic acid aqueous solution is 0.2 mol / L. And / or, the volume ratio of the sodium selenite aqueous solution, the chondroitin sulfate aqueous solution, and the L-ascorbic acid aqueous solution is 1:1:1; And / or, the first stirring treatment is: stirring at room temperature and a speed of 250-350 rpm for 30-50 min; And / or, the second stirring treatment is: stirring at room temperature, in the dark, at a speed of 250-350 rpm for 4 hours; And / or, in the second dialysis treatment, the dialysis bag used has a molecular weight cutoff of 2000-4000 Da, deionized water is used as the dialysate, the dialysis time is 48 h, and the dialysate is changed every 8 h during the dialysis process; And / or, the second freeze-drying process includes a pre-freeze-drying process and a main freeze-drying process performed sequentially; the pre-freeze-drying process is: pre-freezing at -20 ℃ for 24 h, and the main freeze-drying process is: freeze-drying at a vacuum degree ≤10 Pa and a cold trap temperature ≤-50 ℃ for 24 to 48 h.

8. The method for preparing the soluble microneedle patch for the treatment of bacterial keratitis according to claim 4, characterized in that, In step S3, the mass ratio of the crosslinkable matrix to the chondroitin sulfate-selenium nanoparticles is (5-10):1, and the mass of the photoinitiator accounts for 2%-5% of the mass of the crosslinkable matrix. And / or, in step S3, the blending process is as follows: first, stirring at room temperature and 250-350 rpm for 30 min, and then sonicating at room temperature and 100W power for 10-20 min; the sonication adopts an intermittent sonication mode, wherein the intermittent sonication mode is paused for 2 s every 3 s of sonication.

9. The method for preparing the soluble microneedle patch for the treatment of bacterial keratitis according to claim 4, characterized in that, In step S4, the degassing treatment is performed by degassing for 20 to 30 minutes at room temperature and a vacuum degree ≤10 Pa. And / or, in step S4, the photocrosslinking curing is performed at a wavelength of 365 nm and a wavelength of 30 mW / cm². 2 Under light intensity conditions, perform ultraviolet crosslinking curing for 2–3 minutes; And / or, in step S4, the drying process is: drying at 37 °C for 12 h.

10. The application of a soluble microneedle patch in the preparation of a drug for treating bacterial keratitis, characterized in that, The soluble microneedle patch is the soluble microneedle patch for the treatment of bacterial keratitis as described in any one of claims 1 to 3, or the soluble microneedle patch prepared by the preparation method described in any one of claims 4 to 9.

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