Fumaric acid metal organic framework for treating eye germ infection

Abstract

The invention relates to a fumaric acid metal organic framework for treating eye germ infection and application of the fumaric acid metal organic framework in treating eye germ infection. The fumaric acid metal organic framework is Zr / Fe-coated FA MOFs, metal coordination ions are Zr (IV) and Fe (III), and a ligand is fumaric acid. The Zr / Fe-coated FA MOFs has excellent anti-biofilm activity, and the effect of the Zr / Fe-coated FA MOFs can be greatly improved when the Zr / Fe-coated FA MOFs is combined with levofloxacin; besides, the invention further provides a packaged microneedle system Zr / Fe-FA MNs, MOFs can be directly delivered to a bacterial microcolony focus area in a 3D limited microenvironment through the precise penetrating capacity of a microneedle, and the effectiveness of the medicine in biological membrane eradication is remarkably enhanced. Zr / Fe-coated FA MNs not only can overcome the limitation of traditional eye drops in the aspect of cornea permeability, but also can realize long-acting treatment through the sustained release characteristic of MOFs and the sustained release characteristic of microneedles.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceuticals. Specifically, the present invention relates to a metal-organic framework of fumaric acid for treating ocular bacterial infections. Background Art

[0002] Bacterial keratitis is a highly blinding eye disease that mostly occurs in children and young adults, seriously affecting social labor force and quality of life. At present, the number of patients with bacterial keratitis is huge, and with the popularization of contact lens use, the increase in eye trauma and surgeries, the incidence rate is on the rise. Although the research and development of antibiotics have been continuously strengthened in recent years, the speed of the emergence of bacterial drug resistance is much higher than the speed of the research and development of new antibiotics. Clinically, there are problems such as long treatment cycles, poor curative effects, and high recurrence rates. Therefore, the development of new antibacterial drugs and alternative treatment options has become an urgent need in the field of ophthalmology and has great market potential.

[0003] The pathological process of bacterial keratitis begins with the initial adhesion of pathogenic bacteria to the corneal epithelium, and then infiltrates deep into the corneal stroma through the action of invasive enzyme systems. During this process, bacteria form highly organized microcolony structures in the three-dimensional (3D) corneal stromal microenvironment and construct complex biofilms by secreting a large amount of substances such as proteins, polysaccharides, and extracellular DNA. This physical barrier composed of abundant extracellular polymeric substances (EPS) not only provides structural support for bacteria but also significantly enhances their ability to resist host immune defenses. More importantly, the 3D biofilm microenvironment creates a unique ecological niche for bacteria, enabling them to develop multidrug resistance through multiple mechanisms such as regulating the activity of efflux pumps, changing membrane permeability, initiating metabolic adaptation responses, and promoting horizontal gene transfer. Recent studies have shown that the 3D physical microenvironment has a significant mechanobiological regulatory effect on the bacterial colonies / biofilms embedded therein: physical factors such as matrix stiffness, pore structure, and mechanical stress can significantly affect their metabolic activity, gene expression patterns, and drug resistance phenotypes by regulating the quorum sensing system and stress response pathways of bacteria. This 3D microenvironment-dependent drug resistance mechanism poses an insurmountable barrier to traditional antibacterial agents because existing treatment options mainly target the killing effect on planktonic bacteria and ignore the strengthening effect of the 3D biofilm microenvironment on bacterial drug resistance. Therefore, in-depth understanding of the regulatory mechanism of the 3D physical microenvironment on bacterial drug resistance is of great theoretical and clinical significance for the development of new anti-biofilm treatment strategies.

[0004] In the past few decades, research has basically identified three core mechanisms of bacterial drug resistance, including: the formation of an antibiotic molecular penetration barrier, the generation of metabolic heterogeneity, and the activation of adaptive responses to environmental stress and antibiotics. However, it is not only a key regulator of 3D bacterial colony growth and spatial organization, but also profoundly affects bacterial metabolic reprogramming and drug resistance phenotypes through mechanosignal transduction. Specifically, the mechanical properties of the extracellular matrix (ECM) (such as stiffness, topology, and stress distribution) can significantly alter core metabolic pathways such as the tricarboxylic acid cycle (TCA cycle) by regulating bacterial mechanosensitive channels and quorum sensing systems, thereby affecting bacterial antibiotic tolerance and persistence. This discovery provides a new mechanobiological perspective for antibacterial therapy: by precisely regulating the mechanical properties of the ECM, it may be possible to reprogram bacterial metabolism and thus reverse its drug resistance phenotype. This mechanobiology-based antibacterial strategy is significantly innovative: it not only breaks through the single mode of traditional antibiotics directly killing bacteria, but also provides a new treatment paradigm for overcoming antibiotic resistance associated with 3D biofilms by targeting the mechanical-metabolic coupling network of ECM-bacteria. The feasibility of this strategy has been preliminarily verified by experiments. Studies have shown that by regulating the mechanical properties of the ECM, the permeability and killing effect of antibiotics on bacteria in biofilms can be significantly enhanced, laying an important foundation for the development of the next generation of antibacterial treatment regimens.

[0005] Recent studies have shown that the drug resistance of bacteria in a 3D microenvironment is significantly enhanced, and its minimum biofilm eradication concentration (MBEC) can be 2-10,000 times higher than that of planktonic bacteria. The core mechanism of this phenomenon is that the 3D physical microenvironment inhibits the activity of the tricarboxylic acid cycle of bacteria through mechanobiological coupling, resulting in a significant downregulation of bacterial metabolic levels. Metabolic inhibition not only reduces the uptake of antibiotics by bacteria, but also activates multiple drug resistance-related pathways, ultimately leading to a significant reduction in the efficacy of antibiotics.

[0006] Metal-Organic Frameworks (MOFs) are a class of porous crystalline materials formed by the self-assembly of inorganic metal nodes and organic ligands, with unique advantages such as structural designability, degradability, and excellent biocompatibility. MOFs can not only disrupt the structural integrity of bacterial biofilms through physical contact but also induce the generation of reactive oxygen species (ROS) through catalytic activity, thereby exerting a highly efficient anti-biofilm effect. At the same time, as a minimally invasive drug delivery technology, microneedles (MNs) can precisely penetrate the corneal barrier to achieve controlled drug release and show great application potential in the field of ophthalmology.

[0007] In the present invention, the applicant unexpectedly found that MOFs based on zirconium / iron (Zr / Fe) metal centers and fumaric acid (FA) organic ligands have excellent anti-biofilm activity, and their effect can be greatly improved when combined with levofloxacin; the present invention also innovatively combines the Metal-Organic Frameworks (MOFs) with the microneedles (MNs) technology and proposes a new strategy for treating drug-resistant bacterial infectious keratitis based on microneedle delivery of the metabolic regulator Zr / Fe@FA. This strategy not only breaks through the limitations of traditional antibiotic treatments but also provides a practical technical path for the development of new antibacterial regimens based on metabolic regulators, with significant clinical application potential. Summary of the Invention

[0008] Aiming at the problems existing in the prior art, the present invention provides a fumaric acid metal-organic framework, which has excellent anti-microbial effects and can achieve a synergistic anti-microbial effect when combined with levofloxacin.

[0009] In the first aspect, the present invention provides a fumaric acid metal-organic framework, wherein the metal coordination ions of the fumaric acid metal-organic framework are Zr(IV) and Fe(III), and the ligand is fumaric acid.

[0010] In one embodiment, the average particle size of the fumaric acid metal-organic framework is 100 - 300 nm, preferably 100, 150, 200, 250, 300 nm, and more preferably 150 - 250 nm.

[0011] The present invention provides a method for preparing a fumaric acid metal-organic framework, the method comprising: Dissolving a Zr(IV) salt, an Fe(III) salt, and fumaric acid in an organic solvent, reacting at 80 - 140 °C, and collecting the precipitated solid to obtain the fumaric acid metal-organic framework.

[0012] In one embodiment, the Zr(IV) salt is selected from at least one of zirconium tetrachloride, zirconium sulfate, and zirconium nitrate. The Zr(IV) salt can be used in the form of a hydrate, such as zirconium sulfate tetrahydrate, zirconium nitrate trihydrate, zirconium nitrate pentahydrate, etc.

[0013] In one embodiment, the Fe(III) salt is selected from at least one of ferric chloride, ferric sulfate, and ferric nitrate. The Fe(III) salt can be used in the form of a hydrate, such as ferric chloride hexahydrate, ferric nitrate trihydrate, etc.

[0014] In one embodiment, the molar ratio of Zr(IV), Fe(III), and fumaric acid is 0.8 - 1.2:0.8 - 1.2:2.5 - 4.0; preferably, the proportion of Zr(IV) in the molar ratio can be 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, the proportion of Fe(III) in the molar ratio can be 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, 1.1, 1.15, 1.2, and the proportion of fumaric acid in the molar ratio can be 2.5, 3.0, 3.05, 3.1, 3.125, 3.2, 3.25, 3.3, 3.35, 3.4, 3.45, 3.5, 4.0; more preferably, the molar ratio of Zr(IV), Fe(III), and fumaric acid is 1:1:3.0 - 3.3, and even more preferably, the molar ratio of Zr(IV), Fe(III), and fumaric acid is 1:1:3.0 - 3.125.

[0015] In one embodiment, the organic solvent is selected from N,N - dimethylformamide (DMF) and N - methylpyrrolidone (NMP).

[0016] In one embodiment, the amount of the organic solvent used is 50 - 800 ml of the organic solvent per g of fumaric acid, preferably 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800 ml of the organic solvent per g of fumaric acid, and more preferably 250 - 350 ml of the organic solvent per g of fumaric acid.

[0017] In one embodiment, the reaction temperature is 110 - 130 °C, preferably 115 - 125 °C, and more preferably 120 °C.

[0018] In one embodiment, the reaction time is 6 - 48 h, preferably 18 - 36 h, and more preferably 20 - 30 h.

[0019] In one embodiment, the precipitated solid is collected, washed, and dried to obtain the fumaric acid metal - organic framework.

[0020] Preferably, during washing, first wash with the reaction organic solvent, and then wash with absolute ethanol.

[0021] Preferably, dry at 40 - 60 °C.

[0022] In a second aspect, the present invention provides a metal-organic framework of fumaric acid, which is prepared by the said preparation method.

[0023] In a third aspect, the present invention provides a pharmaceutical composition comprising the said metal-organic framework of fumaric acid.

[0024] In one embodiment, the said pharmaceutical composition further comprises other antibacterial drugs.

[0025] Preferably, the said other antibacterial drugs are selected from levofloxacin.

[0026] In the present invention, the mass ratio of the other antibacterial drug to the metal-organic framework of fumaric acid is 1:1000 - 100000, preferably 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:12000, 1:14000, 1:16000, 1:16000, 1:20000, 1:25000, 1:30000, 1:35000, 1:40000, 1:45000, 1:45000, 1:50000, 1:60000, 1:70000, 1:80000, 1:90000, 1:100000, and most preferably, the mass ratio of the other antibacterial drug to the metal-organic framework of fumaric acid is 1:5000 - 40000.

[0027] In a fourth aspect, the present invention provides a pharmaceutical composition comprising fumaric acid and levofloxacin.

[0028] In the said pharmaceutical composition, fumaric acid and levofloxacin are used as drug active ingredients.

[0029] In one embodiment, the mass ratio of levofloxacin to fumaric acid is 1:1000 - 50000, preferably 1:1000, 1:2000, 1:3000, 1:4000, 1:5000, 1:6000, 1:7000, 1:8000, 1:9000, 1:10000, 1:12000, 1:14000, 1:16000, 1:16000, 1:20000, 1:25000, 1:30000, 1:35000, 1:40000, 1:45000, 1:45000, 1:50000, and most preferably, the mass ratio of levofloxacin to the metal-organic framework of fumaric acid is 1:5000 - 20000.

[0030] In a fifth aspect, the present invention further provides the use of the metal-organic framework of fumaric acid or the pharmaceutical composition in the preparation of a drug for antibacterial or anti-biofilm purposes.

[0031] In one embodiment, the drug is used for treating ocular bacterial infections.

[0032] Preferably, the eye part includes the cornea.

[0033] Preferably, the drug is used for treating bacterial keratitis.

[0034] In one embodiment, the drug is used for anti-Staphylococcus aureus.

[0035] In one embodiment, the drug or the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant or carrier.

[0036] In one embodiment, the dosage form of the drug or the pharmaceutical composition includes microneedles, eye drops, local injections, etc., but is not limited thereto.

[0037] In a sixth aspect, the present invention further provides a microneedle patch, comprising a substrate and microneedles, and the microneedles contain the metal-organic framework of fumaric acid.

[0038] In one embodiment, the substrate is made of a polymer.

[0039] Preferably, the polymer is a water-soluble polymer.

[0040] Preferably, the polymer comprises at least one of polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). More preferably, the polymer is polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP). In one embodiment, the substrate further contains hyaluronic acid (HA).

[0041] In one embodiment, the microneedles further contain a polymer.

[0042] Preferably, the polymer is a water-soluble polymer.

[0043] Preferably, the polymer comprises at least one of polyvinyl alcohol (PVA) and polyvinylpyrrolidone (PVP).

[0044] In one embodiment, the content of the metal-organic framework of fumaric acid in the microneedles is 0.05 - 5 wt%, preferably 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 3, 4, 5 wt%, and more preferably 0.1 - 1.5 wt%.

[0045] In one embodiment, other antibacterial agents are also included in the microneedles.

[0046] Preferably, the other antibacterial agent is selected from levofloxacin.

[0047] Preferably, the mass ratio of the other antibacterial agent to the metal-organic framework of fumaric acid is 1:0.1 - 10, preferably 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, and most preferably, the mass ratio of the other antibacterial agent to the metal-organic framework of fumaric acid is 1:0.6 - 1.

[0048] In one embodiment, the diameter of the microneedle patch is 5 - 10 mm, preferably 6 - 8 mm, and more preferably 7 mm.

[0049] In one embodiment, 30 - 100 microneedles are distributed on the microneedle patch, preferably 40 - 70, and more preferably 50 - 60.

[0050] In one embodiment, the height of the microneedles is 200 - 400 μm, preferably 250 - 350 μm, and more preferably 300 μm.

[0051] In one embodiment, the bottom diameter of the microneedles is 100 - 200 μm, preferably 120 - 175 μm, and more preferably 150 μm.

[0052] In one embodiment, the ratio of the height of the microneedles to the bottom diameter is 1.2 - 4:1, preferably 1.2:1, 1.5:1, 1.8:1, 2:1, 2.2:1, 2.5:1, 3:1, 3.5:1, 4:1, and more preferably 1.8 - 2.5:1.

[0053] In a seventh aspect, the present invention also provides a method for preparing the microneedle patch, including: 1. Based on surface projection stereolithography technology, a high-precision 3D printer is used to prepare a microneedle male mold, and then a female mold is obtained by casting with polydimethylsiloxane; 2. The mixed solution containing the metal-organic framework of fumaric acid and the polymer is used as the tip solution. After injecting the tip solution into the mold, the excess solution on the surface is scraped off with a spatula and cured to form the tip structure; 3. The solution containing the polymer is used as the flexible substrate solution. The flexible substrate solution is injected until the mold is full, and after vacuum suction treatment, it is dried and demolded to obtain the microneedle patch.

[0054] In one embodiment, the concentration of the metal-organic framework of fumaric acid in the mixed solution described in step 2 is 0.5 - 50 mg / ml, preferably 0.5, 1, 2, 3, 5, 8, 10, 20, 30, 40, 50 mg / ml, and more preferably 2 - 5 mg / ml.

[0055] In one embodiment, the mixed solution described in step 2 further contains levofloxacin at a concentration of 0.5 - 50 mg / ml. Preferably, the concentration of levofloxacin is 0.5, 1, 1.5, 2, 2.5, 3, 5, 8, 10, 20, 30, 40, 50 mg / ml, and more preferably 2.5 - 5 mg / ml.

[0056] In one embodiment, the total concentration of the polymer in the mixed solution described in step 2 is 20 - 50 wt%, preferably 30 - 40 wt%.

[0057] In one embodiment, the mixed solution described in step 2 contains 10 - 25 wt% of polyvinyl alcohol (PVA) and 10 - 25 wt% of polyvinylpyrrolidone (PVP); preferably, it contains 15 - 20 wt% of polyvinyl alcohol (PVA) and 15 - 20 wt% of polyvinylpyrrolidone (PVP).

[0058] In one embodiment, the solution containing the polymer in step 3 contains 10 - 25 wt% of polyvinyl alcohol (PVA) and 10 - 25 wt% of polyvinylpyrrolidone (PVP); preferably, it contains 15 - 20 wt% of polyvinyl alcohol (PVA) and 15 - 20 wt% of polyvinylpyrrolidone (PVP).

[0059] In one embodiment, the solution containing the polymer in step 3 further contains hyaluronic acid (HA) at a concentration of 0.5 - 3 wt%; preferably, it contains hyaluronic acid (HA) at a concentration of 1 - 2 wt%.

[0060] In one embodiment, the solvent of the solutions described in steps 2 and 3 is water.

[0061] In an eighth aspect, the present invention also provides the use of the microneedle patch in the preparation of a drug for treating germ infections in a 3D confined microenvironment.

[0062] Preferably, the drug is used for treating ocular germ infections.

[0063] Preferably, the eye includes the cornea.

[0064] Preferably, the drug is used for treating bacterial keratitis.

[0065] In one embodiment, the drug is used against Staphylococcus aureus.

[0066] Beneficial effects: The present invention provides a fumaric acid metal-organic framework for treating ocular bacterial infections, and also provides its application in treating ocular bacterial infections. The present invention discovers that MOFs based on zirconium / iron (Zr / Fe) metal centers and fumaric acid (FA) organic ligands have excellent anti-biofilm activity, and their effect can be greatly improved when combined with levofloxacin; in addition, the present invention also innovatively combines MOFs with microneedle technology and designs a microneedle system encapsulated with MOFs based on zirconium / iron (Zr / Fe) metal centers and fumaric acid (FA) organic ligands (abbreviated as Zr / Fe@FA MNs). This delivery system directly delivers MOFs to the bacterial microcolony lesion area in a 3D confined microenvironment through the precise penetration ability of microneedles, significantly enhancing the effectiveness of the drug in biofilm eradication. Zr / Fe@FA MNs can not only overcome the limitations of traditional eye drops in corneal permeability, but also achieve long-term treatment through the sustained release of MOFs and the slow-release characteristics of microneedles. This innovative drug delivery strategy provides an efficient, precise and minimally invasive solution for the treatment of bacterial keratitis, and has broad clinical application prospects. It is expected that this technology will significantly improve the treatment compliance of patients, shorten the treatment course, and reduce the recurrence rate, opening up a new direction for the innovation of ophthalmic drug delivery systems. Description of the drawings

[0067] Figure 1 : Three-dimensional microenvironment culture of bacterial microcolonies and changes in antibiotic resistance. (A) Schematic diagram of the formation of bacterial microcolonies in 3D methacrylated hydrogels. (B) Confocal images of bacterial microcolonies cultured in hydrogels for 1 day, 3 days, and 5 days. C, D Confocal images (C) and quantification results (D) of live / dead staining of microcolonies in 2D and 3D microenvironments after treatment with 0 - 25 μg / mL levofloxacin. Live bacteria and dead bacteria are labeled green and red, respectively. Scale bar: 400 μm. (E) Line graph showing the survival rate of 2D and 3D cultured microcolonies as a function of levofloxacin concentration (0 - 500 μg / mL). (F) Schematic diagram describing that fumarate (FA) enhances the tricarboxylic acid cycle and metabolic activity, thereby regulating the antibiotic resistance of bacterial microcolonies. (G), (H) Confocal images (G) and quantification results (H) of live (green) / dead (red) staining of microcolonies in 3D hydrogels with or without FA after treatment with 0.25 μg / mL, 2.5 μg / mL, and 5 μg / mL levofloxacin. Scale bar: 400 μm. (I) Survival rate of 3D cultured microcolonies treated with 0 - 500 μg / mL levofloxacin with or without FA treatment.

[0068] Figure 2 : Preparation, Physical Characterization, and Antibiofilm Properties of Zr / Fe@FA. (A) Schematic illustration of the preparation process of Zr / Fe@FA. (B) Representative scanning electron microscopy (SEM) image of Zr / Fe@FA. (C) Transmission electron microscopy (TEM) image of Zr / Fe@FA and the corresponding elemental mapping. (D) X-ray photoelectron spectroscopy (XPS) scan of Zr / Fe@FA. (E) Fourier transform infrared spectroscopy (FTIR) of Zr / Fe@FA and FA. (F) X-ray diffraction (XRD) pattern of Zr / Fe@FA after soaking in DPBS for 7 days. G, H Photographs of biofilms stained with crystal violet (CV) (G) and quantification of absorbance of microcolonies at 590 nm after treatment with different concentrations of Zr / Fe@FA and levofloxacin (H). I, J Confocal images of SYTO9 / PI dual-fluorescent stained biofilms (I) and quantification of relative red fluorescence intensity (J) in different treatment groups. Scale bar: 200 µm.

[0069] Figure 3 : Preparation, Physical Characterization, and In Vivo Application of Microneedles. (A) Schematic illustration showing the manufacturing process of bilayer microneedles loaded with Zr / Fe@FA and levofloxacin. (B) Confocal images of microneedles at three different cross-sections: tip, middle, and base. Scale bar: 200 µm. (C) Optical and scanning electron microscopy (SEM) images of microneedles. Scale bar: 200 µm. (D) Optical coherence tomography (OCT) photographs of the cornea before and after application of microneedles. (E) Force-distance curves of microneedles incorporated with LVX (levofloxacin), MOFs (metal-organic frameworks), and MOFs + LVX.

[0070] Figure 4 : In Vivo Evaluation of the Efficacy of Zr / Fe@FA in Reducing Antibiotic Resistance Using a Rabbit Bacterial Keratitis (BK) Model. (A) Overview of the establishment, administration, and evaluation of the BK rabbit model. B, C Anterior segment photographs (B) and OCT images (C) of different treatment groups at 0, 1, 3, 7, and 12 days. (D) Representative images of Staphylococcus aureus colonies on LB agar of different treatment groups. E, F, G Corneal clinical scores (E), central corneal thickness (F), and colony-forming unit (CFU) counts (G) of different treatment groups at 12 days. H, I Corneal H&E staining (H) and Giemsa staining (I) images of different treatment groups at 7 and 12 days. Scale bars: 1 mm and 100 µm.

[0071] In each figure, P < 0.05, P < 0.01, P < 0.001. Detailed implementation mode

[0072] The present invention is described in more detail below to facilitate the understanding of the present invention.

[0073] It should be understood that the terms or words used in the specification and claims should not be construed as having the meanings defined in the dictionary, but should be construed as having meanings consistent with their meanings in the context of the present invention based on the following principles: the concept of the terms can be appropriately defined by the inventor for the best description of the present invention.

[0074] Experimental example 1: Fumaric acid targets to improve 3D bacterial metabolism and reduce drug resistance A 3D bacterial culture model was constructed using GelMA hydrogel to study the formation mechanism and drug resistance characteristics of bacterial biofilms in a confined microenvironment ( Figure 1 A). 1 g of gelatin was completely dissolved in 10 mL of Dulbecco's phosphate buffered saline (DPBS) and heated at 60 °C for 1 hour. Subsequently, 0.8 mL of methacrylic anhydride was added dropwise to the solution, and the reaction was continuously stirred at 50 °C for 3 hours. After the reaction was terminated, the mixture was diluted with 40 mL of DPBS, and the resulting product was dialyzed in deionized water at 55 °C for 5 days through a dialysis membrane with a molecular weight cut-off of 12 - 14 kDa to remove residual methacrylic anhydride. The final solution was filtered and freeze-dried for 12 hours. When preparing the GelMA precursor solution, triethanolamine (TEA, 1.5% w / v), ascorbic acid (VC, 0.1% w / v), and eosin Y (0.1 mM) were dissolved in a 10% w / v GelMA solution, and the prepared GelMA solution was stored at 4 °C for later use.

[0075] The study found that compared with 2D planar culture, bacteria in the 3D culture system showed a unique growth pattern: single planktonic bacteria underwent division and proliferation (day 1), formed in-situ microcolonies (day 3), colony fusion (day 5), and finally formed mature biofilms ( Figure 1 B). To explore the effect of the 3D confined environment on the efficacy of antibiotics, Staphylococcus aureus ATCC 29213 was selected as the model strain, and a susceptibility test was performed with levofloxacin (LVX, MIC = 0.25 μg / mL). The results showed that after treatment with 25 μg / mL LVX for 24 hours, the bacterial mortality rate in the 3D culture system (54.1%) was significantly lower than that in the 2D culture system (82.2%) ( Figure 1 C, D). Further determination found that the minimum biofilm eradication concentration (MBEC = 56.4 μg / mL) of the 3D culture system was significantly higher than that of the 2D system (4.3 μg / mL) ( Figure 1E), indicating that the 3D confined environment can significantly enhance bacterial drug resistance. And 15 mM fumaric acid can significantly enhance the metabolic activity, respiratory function and drug uptake ability of bacteria in the 3D culture system ( Figure 1 F). Under the action of fumaric acid, the bactericidal efficiency of LVX is significantly improved: 5 μg / mL LVX and 0.25 μg / mL LVX + 15 mM fumaric acid have comparable bactericidal effects, and the bactericidal efficiency is increased by 20 times ( Figure 1 G, H). The combination of drugs reduces the MBEC to 2.5 μg / mL ( Figure 1 I), confirming that fumaric acid can be used as an effective antibiotic adjuvant to significantly improve the clearance efficiency of antibiotics against bacterial biofilms in the 3D confined environment.

[0076] Example 1: Preparation of Zr / Fe@FA metal-organic framework nanoparticles ( Figure 2 A) Dissolve 8 mmol (2.16 g) of ferric chloride hexahydrate (FeCl3·6H2O), 8 mmol (1.86 g) of zirconium tetrachloride (ZrCl4) and 2.5 mmol (0.292 g) of fumaric acid (FA) in 30 ml of N,N-dimethylformamide (DMF) respectively. After mixing, continuously stir until completely dissolved. Subsequently, the mixed solution is reacted in a reaction kettle at 120 °C for 24 hours. Discard the supernatant, and the precipitate is washed alternately with DMF and absolute ethanol. After collecting the precipitate, it is dried overnight in an oven at 40 °C, and finally about 200 mg of the target product (Zr / Fe@FA) is obtained.

[0077] Confirmed by SEM, TEM and EDX analysis, the obtained material is nanoparticles with an average particle size of about 221.2 nm, having good crystallinity and uniform element distribution ( Figure 2 B, C). XPS analysis shows the presence of Zr 3d and Fe 2p characteristic peaks in the material. The C=O stretching vibration peak in the FTIR spectrum shifts from 1680 cm⁻¹ to 1656.82 cm⁻¹, and Zr-O and Fe-O characteristic peaks at 653.97 cm⁻¹ and 489 cm⁻¹ appear, confirming the formation of metal-ligand coordination bonds ( Figure 2 D, E). The XRD pattern shows that the material has typical MOFs crystal structure characteristics ( Figure 2 F).

[0078] Test Example 1: Anti-biofilm function of Zr / Fe@FA MOFs To form a biofilm, a concentration of 1×10 8A suspension of Staphylococcus aureus ATCC 29213 bacteria at CFU / mL was inoculated into a 96-well plate and cultured at 37 °C for 72 hours to form a biofilm. Subsequently, different concentrations of metal-organic frameworks (MOFs) and levofloxacin (LVX) were added to the pre-formed biofilm. After discarding the supernatant, the biofilm was stained with 1% crystal violet (CV) for 15 minutes. After washing 3 times with PBS, the biofilm was dissolved with 33% glacial acetic acid, and the absorbance at 590 nm was measured on an enzyme-linked immunosorbent assay (ELISA) reader.

[0079] Compared with the control group, after adding Zr / Fe@FA, the degree of crystal violet staining of the biofilm was significantly reduced. 1 MIC LVX (LVX is levofloxacin, MIC = 0.25 μg / mL) combined with 2 mg / mL Zr / Fe@FA could completely destroy the biofilm, and the effect was equivalent to that of 20 MIC LVX ( Figure 2 G, H). Confocal laser scanning microscopy (CLSM) observations showed that after adding MOFs, the mortality rate of LVX-treated bacterial colonies increased significantly ( Figure 2 I, J), indicating that Zr / Fe@FA could act as an effective antibiotic adjuvant and significantly enhance the destruction of 3D biofilms. Therefore, Zr / Fe@FA MOFs could synergistically exert anti-biofilm and bactericidal effects with LVX.

[0080] Example 2: To solve the problem of poor corneal permeability of drugs and ensure their function in a 3D microenvironment, the present invention developed a microneedle patch for delivering Zr / Fe@FA to the corneal stroma. PVA / PVP was selected as the needle body material to provide sufficient strength and toughness respectively; hyaluronic acid (HA) was added to the substrate to enhance the moisturizing performance.

[0081] The specific preparation method is as Figure 3 shown in A: The tip of the microneedle patch was conical, and the aspect ratio was 2:1 (height 300 µm, bottom diameter 150 µm). First, based on surface projection stereolithography technology, a high-precision 3D printer (S230) was used to prepare a microneedle male mold, and then a female mold was obtained by casting with polydimethylsiloxane (PDMS). The microneedles were prepared using a two-step micromolding process: The first step: A drug solution containing LVX at a final concentration of 5 mg / mL, MOFs at 2 mg / mL, or 2 mg / mL MOFs + 2.5 mg / mL LVX and a mixed solution of PVA at a final concentration of 20 wt% and PVP at 20 wt% (the solvent was ultrapure water) was used as the tip solution, and a mixed solution containing PVA at a final concentration of 20 wt%, PVP at 20 wt%, and hyaluronic acid (HA) at 1 wt% (the solvent was ultrapure water) was used as the flexible substrate solution. After injecting the tip solution into the mold, the excess solution on the surface was scraped off with a spatula, and the tip structure was formed by curing at room temperature for 1 hour.

[0082] Step 2: Inject the flexible substrate solution until the mold is full. After vacuum suction treatment, place it in an environment at 30 °C and dry for 10 hours. After demolding, the finished microneedles are stored in a refrigerator at 4 °C for later use.

[0083] The microneedle patch has a diameter of 7 mm, which matches the corneal area. Each patch is evenly distributed with about 50 needles. The tip of the needle is conical, and the aspect ratio is 2:1 (height 300 µm, bottom diameter 150 µm) ( Figure 3 B). The results of SEM and fluorescence microscopy show that the microneedles have a good layered structure, and the needle height is 300 µm ( Figure 3 B, C). OCT observation shows that after the microneedle patch adheres to the cornea, Zr / Fe@FA can be minimally invasively dissolved and penetrate into the deep layer of the cornea without leaving obvious traces ( Figure 3 D). Mechanical tests show that although the addition of MOFs reduces the strength of the microneedles, it still exceeds the minimum skin puncture force (~3 Mpa), ensuring that it can penetrate the cornea ( Figure 3 E).

[0084] Experimental Example 2: Efficient clearance of 3D corneal stromal bacterial microcolonies by microneedle delivery of Zr / Fe@FA and antibiotics A 3D corneal stromal infection model was established by injecting 100 μl of a 1×105 CFU / mL Staphylococcus aureus ATCC 29213 bacterial suspension into the superficial stromal layer of the rabbit cornea to evaluate the effect of Zr / Fe@FA on enhancing the bactericidal effect of antibiotics ( Figure 4 A). The rabbits with bacterial keratitis models were randomly divided into six groups: control group (no treatment), MN:MOFs+LVX group, MN:LVX group, MN:MOFs group, ED:MOFs+LVX group, and ED:LVX group. The MN group was given microneedle patches at intervals of 6 hours on days 0, 1, 2, and 4, and the ED group was given an equal amount of eye drops. Slit lamp and AS-OCT observations were performed on days 0, 1, 3, 7, and 12, and the animals were sacrificed on days 3, 7, and 10 for bacterial culture counting and histopathological analysis ( Figure 4 B). The results showed that 24 hours after inoculating bacteria (recorded as day 0), a circular infiltration with a diameter of 5 mm formed in the center of the cornea, accompanied by conjunctival congestion and a large amount of secretions, and a BK model was successfully established. On day 1, except for the MN:MOFs+LVX group showing local infiltration with reduced secretions, extensive infiltration and aggravated infection occurred in the other groups. On day 7, the infection focus in the MN:MOFs+LVX group basically disappeared, and only mild corneal edema was seen; on day 12, the cornea was completely transparent or only left a superficial scar. In contrast, corneal opacity and hypopyon occurred in other groups, and corneal neovascularization was also observed in the control group and the ED:LVX group. Figure 4B). Corneal fluorescein staining showed that in the MN:MOFs + LVX group, the stained area decreased over time, the epithelial repair rate was faster, and the clinical score was lower ( Figure 4 E). AS-OCT observation showed that in the control group, corneal infiltration (increased gray value) was accompanied by anterior chamber exudation, and bullous keratopathy occurred on the 12th day; while in the MN:MOFs + LVX group, the corneal infiltration area gradually decreased, and the corneal epithelium was intact and the thickness became thinner on the 7th and 12th days ( Figure 4 C, F). Bacterial culture count showed that on the 3rd day, the bacterial load in each group decreased compared with the control group, and the antibacterial effect of the MN group was more significant ( Figure 4 D). After 12 days of treatment, no bacteria were detected in the MN:MOFs + LVX group, and the antibacterial effect was the best ( Figure 4 G).

[0085] Histopathological analysis showed that on the 7th and 12th days, significant inflammatory cell infiltration occurred in the control group, MN:MOFs group, MN:LVX group, ED:MOFs + LVX group and ED:LVX group, while the inflammatory cells in the MN:MOFs + LVX group were significantly reduced ( Figure 4 H). Masson staining showed that the collagen in the MN:MOFs + LVX group was arranged neatly, while the collagen in other groups was disordered and the structure was changed ( Figure 4 I). The results of Giemsa staining were consistent with the bacterial culture. Almost no bacteria were seen in the MN:MOFs + LVX group on the 7th and 12th days ( Figure 4 J).

[0086] The preferred embodiments of the present invention are described above, but they are not intended to limit the present invention. Those skilled in the art can make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A metal-organic framework of fumaric acid for treating ocular bacterial infections, wherein the metal coordination ions of the metal-organic framework of fumaric acid are Zr(IV) and Fe(III), and the ligand is fumaric acid.

2. The metal-organic framework of fumaric acid according to claim 1, wherein The average particle size of the metal-organic framework of fumaric acid is 100 - 300 nm, preferably 150 - 250 nm.

3. The preparation method of the metal-organic framework of fumaric acid according to claim 1 or 2, characterized in that, The preparation method of the metal-organic framework of fumaric acid comprises: Dissolving a Zr(IV) salt, an Fe(III) salt and fumaric acid in an organic solvent, heating to react at 80 - 140 °C, and collecting the precipitated solid to obtain the metal-organic framework of fumaric acid.

4. The preparation method according to claim 3, wherein The molar ratio of Zr(IV), Fe(III), and fumaric acid is 0.8 - 1.2:0.8 - 1.2:2.5 - 4.0; the organic solvent is selected from N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP); the reaction temperature is 110 - 130 °C.

5. A metal-organic framework of fumaric acid prepared by the preparation method according to claim 3 or 4.

6. A pharmaceutical composition comprising the metal-organic framework of fumaric acid according to any one of claims 1, 2, and 5.

7. The pharmaceutical composition according to claim 6, wherein The pharmaceutical composition further comprises other antibacterial drugs, and the other antibacterial drugs are selected from levofloxacin.

8. A pharmaceutical composition comprising fumaric acid and levofloxacin.

9. Use of the metal-organic framework of fumaric acid according to any one of claims 1, 2, and 5 or the pharmaceutical composition according to any one of claims 6 - 8 in the preparation of a drug for antibacterial or anti-biofilm purposes.

10. The application according to claim 9, characterized in that, The drug is used for treating ocular bacterial infections.

11. A microneedle patch comprising a substrate and microneedles, wherein the microneedles contain the metal-organic framework of fumaric acid according to any one of claims 1, 2, and 5.

Images