A fumarate metal-organic framework for treating eye bacterial infections

Abstract

The present invention relates to a fumarate metal-organic framework for treating ocular bacterial infections, and also relates to its use in treating ocular bacterial infections. The fumarate metal-organic framework is Zr / Fe@FA MOFs, the metal coordination ions are Zr(IV) and Fe(III), and the ligand is fumaric acid. The Zr / Fe@FA MOFs have excellent anti-biofilm activity, and their effect can be greatly improved when used in combination with levofloxacin. In addition, the present invention also provides an encapsulated microneedle system, Zr / Fe@FA MNs, which can deliver MOFs directly to bacterial microcolony lesion areas in 3D confined microenvironments through the precise penetration ability of microneedles, significantly enhancing the effectiveness of drugs 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 properties of microneedles.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a fumarate metal organic framework for treating eye bacterial infection. Background Art

[0002] Bacterial keratitis is a highly blinding eye disease that often affects children and young adults, severely impacting society's workforce and quality of life. Currently, the number of people suffering from bacterial keratitis is enormous, and with the increasing use of contact lenses, eye trauma, and surgeries, the incidence rate is on the rise. Despite the continuous increase in antibiotic research and development efforts in recent years, the rate of bacterial resistance is far higher than the rate of new antibiotic development, leading to clinical problems such as long treatment cycles, poor efficacy, and high recurrence rates. Therefore, the development of new antimicrobial drugs and alternative treatment options has become an urgent need in the field of ophthalmology, with enormous market potential.

[0003] The pathological process of bacterial keratitis begins with the initial adhesion of pathogens to the corneal epithelium, followed by infiltration into the deep corneal stroma through the action of invasive enzymes. During this process, bacteria form highly organized microcolonies within the three-dimensional (3D) corneal stromal microenvironment and construct complex biofilms by secreting abundant proteins, polysaccharides, and extracellular DNA. This physical barrier, composed of abundant extracellular polymeric substances (EPS), not only provides structural support for the bacteria but also significantly enhances their ability to circumvent host immune defenses. More importantly, the 3D biofilm microenvironment creates a unique ecological niche for bacteria, enabling them to develop multidrug resistance through various mechanisms, including regulating efflux pump activity, altering membrane permeability, initiating metabolic adaptations, 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 the metabolic activity, gene expression patterns, and drug resistance phenotypes of bacteria by regulating their quorum sensing system and stress response pathways. This 3D microenvironment-dependent drug resistance mechanism poses a difficult barrier to overcome for traditional antimicrobial agents, as existing treatment options mainly target the killing effect of planktonic bacteria while ignoring the strengthening effect of the 3D biofilm microenvironment on bacterial resistance. Therefore, a deep understanding of the regulatory mechanism of 3D physical microenvironment on bacterial resistance has important theoretical and clinical significance for the development of new anti-biofilm treatment strategies.

[0004] Over the past few decades, research has essentially identified three core mechanisms of bacterial resistance, including the formation of a permeability barrier for antibiotic molecules, the generation of metabolic heterogeneity, and the activation of adaptive responses to environmental stress and antibiotics. However, bacterial resistance is not only a key regulator of 3D bacterial colony growth and spatial organization, but also profoundly influences bacterial metabolic reprogramming and drug-resistant phenotypes through mechanical signal 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 antimicrobial therapy: by precisely manipulating the mechanical properties of the ECM, it may be possible to reprogram bacterial metabolism and reverse its drug-resistant phenotype. This mechanobiology-based antimicrobial strategy is significantly innovative: it not only transcends the single mode of direct bacterial killing by traditional antibiotics, but also, by targeting the mechanical-metabolic coupling network between ECM and bacteria, offers a new therapeutic paradigm for overcoming antibiotic resistance associated with 3D biofilms. The feasibility of this strategy has been initially validated by experimental studies, demonstrating that modulating the mechanical properties of the ECM can significantly enhance the permeability and killing effect of antibiotics on bacteria within biofilms, laying an important foundation for the development of next-generation antimicrobial treatments.

[0005] Recent studies have shown that bacterial resistance in 3D microenvironments is significantly enhanced, with the minimum biofilm eradication concentration (MBEC) increasing by 2-10,000 times compared to planktonic bacteria. The core mechanism of this phenomenon is that the 3D physical microenvironment inhibits bacterial tricarboxylic acid cycle activity through mechanical-biological coupling, leading to a significant downregulation of bacterial metabolism. This metabolic inhibition not only reduces bacterial antibiotic uptake but also activates multiple resistance-related pathways, ultimately significantly reducing antibiotic efficacy.

[0006] Metal-organic frameworks (MOFs) are a class of porous crystalline materials formed by the self-assembly of inorganic metal nodes and organic ligands. They offer unique advantages such as structural designability, biodegradability, and excellent biocompatibility. MOFs 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 effective anti-biofilm effect. Furthermore, microneedles (MNs), as a minimally invasive drug delivery technology, can precisely penetrate the corneal barrier and achieve controlled drug release, demonstrating significant potential for application in ophthalmology.

[0007] In this invention, the applicant unexpectedly discovered that MOFs based on zirconium / iron (Zr / Fe) metal centers and fumaric acid (FA) organic ligands exhibit excellent anti-biofilm activity, and their combination with levofloxacin significantly enhances its efficacy. The present invention also innovatively combines metal-organic frameworks (MOFs) with microneedle (MN) technology to propose a novel strategy for treating drug-resistant bacterial keratitis by microneedle-delivered metabolic regulators, Zr / Fe@FA. This strategy not only overcomes the limitations of traditional antibiotic treatments but also provides a practical technical path for the development of novel antibacterial regimens based on metabolic regulators, with significant potential for clinical application. Summary of the Invention

[0008] In response to the problems existing in the prior art, the present invention provides a fumarate metal-organic framework, which has excellent antimicrobial effects and can achieve a synergistic antimicrobial effect when used in combination with levofloxacin.

[0009] In a first aspect, the present invention provides a fumarate metal organic framework, wherein the metal coordination ions of the fumarate 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 fumarate 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 fumarate metal organic framework, the method comprising:

[0012] Zr(IV) salt, Fe(III) salt and fumaric acid are dissolved in an organic solvent, heated to 80-140° C. for reaction, and the precipitated precipitate is collected to obtain the fumarate metal organic framework.

[0013] 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, and the like.

[0014] 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, and the like.

[0015] 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 ratio 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, or 1.2, and the ratio of Fe(III) in the molar ratio can be 0.8, 0.85, 0.9, 0.95, 1.0, 1.05, or 1.1 , 1.15, 1.2, 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; also preferably, the molar ratio of Zr(IV), Fe(III), and fumaric acid is 1:1:3.0-3.3, more preferably, the molar ratio of Zr(IV), Fe(III), and fumaric acid is 1:1:3.0-3.125.

[0016] In one embodiment, the organic solvent is selected from N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP).

[0017] In one embodiment, the amount of the organic solvent is 50-800 ml of organic solvent per gram of fumaric acid, preferably, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800 ml of organic solvent per gram of fumaric acid, more preferably, 250-350 ml of organic solvent per gram of fumaric acid.

[0018] In one embodiment, the reaction temperature is 110-130°C, preferably 115-125°C, more preferably 120°C.

[0019] In one embodiment, the reaction time is 6-48 h, preferably 18-36 h, more preferably 20-30 h.

[0020] In one embodiment, the precipitated precipitate is collected, washed, and dried to obtain the fumarate metal organic framework.

[0021] Preferably, the organic solvent used in the reaction is first used for washing, and then anhydrous ethanol is used for washing.

[0022] Preferably, the drying temperature is 40-60°C.

[0023] In a second aspect, the present invention provides a fumarate metal organic framework, which is prepared by the preparation method.

[0024] In a third aspect, the present invention provides a pharmaceutical composition comprising the fumarate metal-organic framework.

[0025] In one embodiment, the pharmaceutical composition further comprises other antibacterial drugs.

[0026] Preferably, the other antibacterial drugs are selected from levofloxacin.

[0027] In the present invention, the mass ratio of other antibacterial drugs to fumarate metal organic framework 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 other antibacterial drugs to fumarate metal organic framework is 1:5000-40000.

[0028] In a fourth aspect, the present invention provides a pharmaceutical composition comprising fumaric acid and levofloxacin.

[0029] In the pharmaceutical composition, fumaric acid and levofloxacin serve as pharmaceutical active ingredients.

[0030] 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, most preferably, the mass ratio of levofloxacin to fumaric acid metal organic framework is 1:5000-20000.

[0031] In a fifth aspect, the present invention further provides use of the fumarate metal organic framework or the pharmaceutical composition in preparing a drug, wherein the drug is used for antibacterial or anti-biofilm.

[0032] In one embodiment, the medicament is for treating bacterial infection of the eye.

[0033] Preferably, the eye includes the cornea.

[0034] Preferably, the medicament is used to treat bacterial keratitis.

[0035] In one embodiment, the medicament is for use against Staphylococcus aureus.

[0036] In one embodiment, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant or carrier.

[0037] In one embodiment, the dosage form of the drug or pharmaceutical composition includes microneedles, eye drops, local injections, etc., but is not limited thereto.

[0038] In a sixth aspect, the present invention further provides a microneedle patch comprising a substrate and microneedles, wherein the microneedles contain the fumarate metal organic framework.

[0039] In one embodiment, the substrate is made of a polymer.

[0040] Preferably, the polymer is a water-soluble polymer.

[0041] Preferably, the polymer comprises at least one of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP). More preferably, the polymer is polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP).

[0042] In one embodiment, the substrate further comprises hyaluronic acid (HA).

[0043] In one embodiment, the microneedles further comprise a polymer.

[0044] Preferably, the polymer is a water-soluble polymer.

[0045] Preferably, the polymer comprises at least one of polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP).

[0046] In one embodiment, the content of the fumarate metal organic framework in the microneedle is 0.05-5wt%, preferably 0.05, 0.1, 0.2, 0.5, 0.8, 1, 1.5, 2, 3, 4, 5wt%, and more preferably 0.1-1.5wt%.

[0047] In one embodiment, the microneedles further contain other antibacterial drugs.

[0048] Preferably, the other antibacterial drugs are selected from levofloxacin.

[0049] Preferably, the mass ratio of other antibacterial drugs to fumarate metal organic framework 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 other antibacterial drugs to fumarate metal organic framework is 1:0.6-1.

[0050] In one embodiment, the diameter of the microneedle patch is 5-10 mm, preferably 6-8 mm, and more preferably 7 mm.

[0051] In one embodiment, the microneedle patch is distributed with 30-100 microneedles, preferably 40-70 microneedles, and more preferably 50-60 microneedles.

[0052] In one embodiment, the height of the microneedles is 200-400 μm, preferably 250-350 μm, and more preferably 300 μm.

[0053] In one embodiment, the diameter of the bottom of the microneedle is 100-200 μm, preferably 120-175 μm, more preferably 150 μm.

[0054] In one embodiment, the ratio of the height to the bottom diameter of the microneedle 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.

[0055] In a seventh aspect, the present invention further provides a method for preparing the microneedle patch, comprising:

[0056] 1. Based on surface projection stereolithography technology, a high-precision 3D printer was used to prepare a positive microneedle mold, and then polydimethylsiloxane was used for casting to obtain a negative mold;

[0057] 2. A mixed solution containing the fumarate metal organic framework and a polymer is used as a needle tip solution. After the needle tip solution is injected into the mold, excess solution on the surface is scraped off with a scraper, and the needle tip structure is obtained by solidification molding;

[0058] 3. A solution containing a polymer is used as a flexible substrate solution, and the flexible substrate solution is injected into the full mold. After vacuum suction treatment, the mold is dried and demolded to obtain a microneedle patch.

[0059] In one embodiment, the concentration of the fumarate metal organic framework in the mixed solution of 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.

[0060] In one embodiment, the mixed solution in step 2 further comprises 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, more preferably 2.5-5 mg / ml.

[0061] In one embodiment, the total concentration of the polymer in the mixed solution in step 2 is 20-50 wt %, preferably 30-40 wt %.

[0062] In one embodiment, the mixed solution in step 2 contains polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) at a concentration of 10-25 wt %; preferably, the mixed solution contains polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) at a concentration of 15-20 wt %.

[0063] In one embodiment, the polymer solution in step 3 contains polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) at a concentration of 10-25 wt %; preferably, contains polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) at a concentration of 15-20 wt %.

[0064] 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, the solution contains hyaluronic acid (HA) at a concentration of 1-2 wt %.

[0065] In one embodiment, the solvent of the solution in step 2 and step 3 is water.

[0066] In an eighth aspect, the present invention further provides a use of the microneedle patch in the preparation of a drug for treating bacterial infection in a 3D confined microenvironment.

[0067] Preferably, the drug is used to treat eye bacterial infections.

[0068] Preferably, the eye includes the cornea.

[0069] Preferably, the medicament is used to treat bacterial keratitis.

[0070] In one embodiment, the medicament is for use against Staphylococcus aureus.

[0071] Beneficial effects:

[0072] This invention provides a fumarate metal-organic framework (MOF) for treating ocular bacterial infections and its use in this treatment. The authors discovered that MOFs based on a zirconium / iron (Zr / Fe) metal center and fumaric acid (FA) organic ligands exhibit excellent anti-biofilm activity, significantly enhancing their efficacy when used in combination with levofloxacin. Furthermore, the authors innovatively combined MOFs with microneedle technology to design a MOF-encapsulated microneedle system (Zr / Fe@FA MNs) based on a Zr / Fe metal center and fumaric acid (FA) organic ligands. This delivery system leverages the precise penetration capabilities of the microneedles to deliver MOFs directly to bacterial microcolony lesions within a 3D confined microenvironment, significantly enhancing the drug's effectiveness in eradicating biofilms. Zr / Fe@FA MNs not only overcome the corneal penetration limitations of traditional eye drops but also achieve long-term therapeutic efficacy through the sustained release properties of the MOFs and the sustained release characteristics of the microneedles. This innovative drug delivery strategy offers an efficient, precise, and minimally invasive solution for the treatment of bacterial keratitis, with broad clinical application prospects. It is expected that this technology will significantly improve patient treatment compliance, shorten treatment duration, and reduce recurrence rates, opening up new avenues for innovation in ophthalmic drug delivery systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1: Three-dimensional microenvironmental culture of bacterial microcolonies and changes in antibiotic resistance. (A) Schematic diagram of bacterial microcolony formation in 3D methacrylated hydrogels. (B) Confocal images of bacterial microcolonies cultured in hydrogels for 1, 3, and 5 days. C, D Confocal images (C) and quantification (D) of live / dead staining of microcolonies in 2D and 3D microenvironments after treatment with 0–25 µg / mL levofloxacin. Live and dead bacteria are marked in green and red, respectively. Scale bar: 400 µm. (E) Discounted plot of the survival rate of 2D and 3D cultured microcolonies as a function of levofloxacin concentration (0–500 µg / mL). (F) Schematic diagram depicting how fumarate (FA) enhances tricarboxylic acid cycle and metabolic activity, thereby regulating antibiotic resistance in bacterial microcolonies. (G), (H) Confocal images (G) and quantification (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 and left untreated with FA.

[0074] Figure 2 Preparation, physical characterization, and antibiofilm properties of Zr / Fe@FA. (A) Schematic diagram of the Zr / Fe@FA preparation process. (B) Typical scanning electron microscopy (SEM) image of Zr / Fe@FA. (C) Transmission electron microscopy (TEM) image of Zr / Fe@FA and corresponding elemental mapping. (D) X-ray photoelectron spectroscopy (XPS) scan of Zr / Fe@FA. (E) Fourier transform infrared (FTIR) spectra of Zr / Fe@FA and FA. (F) X-ray diffraction (XRD) pattern of Zr / Fe@FA after immersion in DPBS for 7 days. G, H Photographs of biofilms stained with crystal violet (CV) (G) and absorbance quantification of microcolonies at 590 nm after treatment with different concentrations of Zr / Fe@FA and levofloxacin (H). I, J Confocal images of biofilms stained with SYTO9 / PI dual fluorescence (I) and quantification of relative red fluorescence intensity (J) in different treatment groups. Scale bar: 200 µm.

[0075] Figure 3Preparation, physical characterization, and in vivo application of microneedles. (A) Schematic diagram showing the fabrication process of bilayer microneedles loaded with Zr / Fe@FA and levofloxacin. (B) Confocal images of the microneedle at three different cross-sections: tip, middle, and base. Scale bar: 200 μm. (C) Optical and scanning electron microscopy (SEM) images of the microneedle. Scale bar: 200 μm. (D) Optical coherence tomography (OCT) images of the cornea before and after microneedle application. (E) Force-distance curves of microneedles incorporating LVX (levofloxacin), MOFs (metal-organic frameworks), and MOFs + LVX.

[0076] Figure 4 The efficacy of Zr / Fe@FA in reducing antibiotic resistance was evaluated in vivo using a rabbit bacterial keratitis (BK) model. (A) Schematic overview of the BK rabbit model establishment, drug administration, and evaluation. B, C: Photographs (B) and optical coherence tomography (OCT) images of the anterior cornea of the different treatment groups after 0, 1, 3, 7, and 12 days. (D) Representative images of Staphylococcus aureus colonies on LB agar in the different treatment groups. E, F, G: Corneal clinical scores (E), central corneal thickness (F), and colony-forming unit (CFU) counts (G) in the different treatment groups after 12 days. H, I: H&E (H) and Giemsa (I) images of the corneas of the different treatment groups after 7 and 12 days. Scale bars: 1 mm and 100 μm.

[0077] In each figure, P<0.05, P<0.01, P<0.001. DETAILED DESCRIPTION

[0078] The present invention is described in more detail below to facilitate understanding of the present invention.

[0079] It should be understood that the terms or words used in the specification and claims should not be understood as having the meaning defined in the dictionary, but should be understood as having the meaning consistent with their meaning in the context of the present invention based on the following principle: the concept of the term can be appropriately defined by the inventor for the best explanation of the present invention.

[0080] Experimental Example 1: Fumaric acid targets and improves 3D bacterial metabolism and reduces drug resistance

[0081] GelMA hydrogel was used to construct a 3D bacterial culture model to study the formation mechanism and drug resistance characteristics of bacterial biofilms in confined microenvironments ( Figure 1A). 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 stirred continuously at 50°C for 3 hours. After the reaction was terminated, the mixture was diluted with 40 mL of DPBS. The resulting product was dialyzed against deionized water at 55°C for 5 days using a dialysis membrane with a molecular weight cutoff of 12-14 kDa to remove residual methacrylic anhydride. The final solution was filtered and freeze-dried for 12 hours. To prepare 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. The prepared GelMA solution was stored at 4°C until use.

[0082] The study found that compared with 2D planar culture, bacteria in 3D culture systems exhibited a unique growth pattern: after being embedded in the hydrogel, single planktonic bacteria underwent division and proliferation (day 1), formed in situ microcolonies (day 3), colony fusion (day 5), and finally formed a mature biofilm (day 6). Figure 1 B). To investigate the effect of a 3D confined environment on antibiotic efficacy, Staphylococcus aureus ATCC 29213 was selected as a model strain, and a drug susceptibility test was conducted using levofloxacin (LVX, MIC = 0.25 μg / mL). The results showed that after 24 hours of treatment with 25 μg / mL LVX, 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 measurements revealed that the minimum biofilm elimination 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 1 E), indicating that 3D confined environment can significantly enhance bacterial resistance. 15mM fumaric acid can significantly enhance the metabolic activity, respiratory function and drug uptake ability of bacteria in 3D culture system ( Figure 1 F). Under the action of fumaric acid, the bactericidal efficiency of LVX is significantly improved: 5μg / mL LVX and 0.25μg / mL LVX+15mM fumaric acid have comparable bactericidal effects, and the bactericidal efficiency is increased by 20 times ( Figure 1 G, H). Combined use of the drug reduced MBEC to 2.5 μg / mL ( Figure 1 I) confirmed that fumaric acid can be used as an effective antibiotic adjuvant, significantly improving the efficiency of antibiotics in clearing bacterial biofilms in 3D confined environments.

[0083] Example 1: Preparation of Zr / Fe@FA metal organic framework nanoparticles ( Figure 2 A)

[0084] 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) were dissolved in 30 ml of N,N-dimethylformamide (DMF), mixed, and stirred continuously until completely dissolved. The mixture was then reacted in an autoclave at 120°C for 24 hours. The supernatant was discarded, and the precipitate was washed alternately with DMF and anhydrous ethanol. The precipitate was collected and dried in an oven at 40°C overnight, yielding approximately 200 mg of the desired product (Zr / Fe@FA).

[0085] SEM, TEM, and EDX analyses confirmed that the obtained material was nanoparticles with an average particle size of approximately 221.2 nm, 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 the Zr-O and Fe-O characteristic peaks appear at 653.97 cm⁻¹ and 489 cm⁻¹, confirming the formation of metal-ligand coordination bonds ( Figure 2 D, E). XRD patterns show that the material has typical MOFs crystal structure characteristics ( Figure 2 F).

[0086] Experimental Example 1: Anti-biofilm function of Zr / Fe@FA MOFs

[0087] In order to form a biofilm, the concentration of 1×10 8 A suspension of Staphylococcus aureus ATCC 29213 at 500 CFU / mL was inoculated into a 96-well plate and incubated at 37°C for 72 hours to form a biofilm. Subsequently, various concentrations of metal-organic frameworks (MOFs) and levofloxacin (LVX) were added to the preformed biofilm. After the supernatant was discarded, the biofilm was stained with 1% crystal violet (CV) for 15 minutes. After washing three times with PBS, the biofilm was solubilized with 33% glacial acetic acid, and the absorbance at 590 nm was measured on a microplate reader.

[0088] Compared with the control group, the crystal violet staining of the biofilm was significantly reduced after the addition of Zr / Fe@FA. 1 MIC LVX (LVX is levofloxacin, MIC = 0.25 μg / mL) combined with 2 mg / mL Zr / Fe@FA can completely destroy the biofilm, and the effect is equivalent to 20 MIC LVX ( Figure 2 G, H). CLSM observations showed that the mortality rate of bacterial colonies treated with LVX was significantly increased after the addition of MOFs ( Figure 2Figures I, J indicate that Zr / Fe@FA can act as an effective antibiotic adjuvant, significantly enhancing the disruption of 3D biofilms. Therefore, Zr / Fe@FA MOFs can synergize with LVX in anti-biofilm and bactericidal effects.

[0089] Example 2:

[0090] To address the problem of poor corneal drug penetration and ensure its efficacy within a 3D microenvironment, the researchers developed a microneedle patch for delivering Zr / Fe@FA to the corneal stroma. PVA / PVP were used as the needle body materials, providing sufficient strength and toughness, respectively; hyaluronic acid (HA) was added to the base to enhance moisturizing properties.

[0091] Specific preparation method Figure 3 As shown in Figure A: The microneedle patch has a conical tip with an aspect ratio of 2:1 (300 µm height, 150 µm base diameter). First, a high-precision 3D printer (S230) based on surface projection stereolithography was used to create a positive microneedle mold. Then, polydimethylsiloxane (PDMS) was used to cast the negative mold. The microneedles were fabricated using a two-step micromolding process:

[0092] Step 1: A mixture of drugs (drugs at final concentrations of 5 mg / mL LVX, 2 mg / mL MOFs, or 2 mg / mL MOFs plus 2.5 mg / mL LVX) and a final concentration of 20 wt% PVA and 20 wt% PVP (in ultrapure water) was used as the tip solution. A mixture of PVA, 20 wt% PVP, and 1 wt% hyaluronic acid (HA) (in ultrapure water) was used as the flexible substrate solution. After the tip solution was injected into the mold, excess solution was scraped off the surface with a spatula, and the mold was cured at room temperature for 1 hour to form the tip structure.

[0093] Step 2: Fill the mold with the flexible substrate solution, vacuum it, and dry it at 30°C for 10 hours. After demolding, store the finished microneedles in a refrigerator at 4°C until ready for use.

[0094] The microneedle patch has a diameter of 7 mm, matching the area of the cornea. Each patch has approximately 50 needles evenly distributed. The needle tips are conical with an aspect ratio of 2:1 (300 µm in height and 150 µm in bottom diameter). Figure 3 B). SEM and fluorescence microscopy results show that the microneedles have a good hierarchical structure and the needle height is 300 μm ( Figure 3 B, C). OCT observations showed that after the microneedle patch was attached to the cornea, Zr / Fe@FA could be minimally invasively dissolved and penetrated into the deep layers of the cornea without leaving any obvious traces ( Figure 3D). Mechanical tests showed that although the addition of MOFs reduced the strength of the microneedle, it still exceeded the minimum puncture force of the skin (~3 Mpa), ensuring that it can penetrate the cornea ( Figure 3 E).

[0095] Experimental Example 2: Microneedle delivery of Zr / Fe@FA and antibiotics can effectively eliminate 3D corneal stromal bacterial microcolonies

[0096] A 3D corneal stromal infection model was established by injecting 100 μl of 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 in enhancing the bactericidal effect of antibiotics. Figure 4 A). Rabbits with a bacterial keratitis model 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 6-hour intervals on days 0, 1, 2, and 4, while 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, respectively. The animals were sacrificed on days 3, 7, and 10 for bacterial culture counts and histopathological analysis ( Figure 4 B). The results showed that 24 hours after bacterial inoculation (recorded as day 0), a circular infiltration with a diameter of 5 mm was formed in the center of the cornea, accompanied by conjunctival congestion and a large amount of secretions, and the BK model was successfully established. On day 1, except for the MN:MOFs+LVX group, which showed local infiltration with reduced secretions, the other groups showed extensive infiltration and worsening infection. 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 shallow scars remained. In contrast, the other groups developed corneal opacity and anterior chamber abscess, and corneal neovascularization was also observed in the control group and ED:LVX group ( Figure 4 B). Corneal fluorescein staining showed that the staining area of the MN:MOFs+LVX group decreased over time, the epithelial repair rate was faster, and the clinical score was lower ( Figure 4 E). AS-OCT observation showed that the control group had corneal infiltration (increased grayscale value) with anterior chamber exudation, and bullous keratopathy appeared on the 12th day; while the MN:MOFs+LVX group had corneal infiltration area gradually reduced, and the corneal epithelium was intact and thinner on the 7th and 12th days ( Figure 4 C, F). Bacterial culture counts showed that the bacterial load in each group decreased compared with the control group on the third day, 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, indicating the best antibacterial effect ( Figure 4 G).

[0097] Histopathological analysis showed that on days 7 and 12, significant inflammatory cell infiltration was observed in the control group, MN:MOFs group, MN:LVX group, ED:MOFs+LVX group, and ED:LVX group, while the number of inflammatory cells in the MN:MOFs+LVX group was significantly reduced ( Figure 4 H). Masson staining showed that the collagen in the MN:MOFs+LVX group was neatly arranged, while the collagen in other groups was disordered and structurally altered ( Figure 4 I). The Giemsa staining results were consistent with the bacterial culture results. Almost no bacteria were found in the MN:MOFs+LVX group on days 7 and 12 ( Figure 4 J).

[0098] 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 may make improvements and changes to the embodiments disclosed herein without departing from the scope and spirit of the present invention.

Claims

1. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises a fumarate metal-organic framework and other antibacterial drugs, wherein the other antibacterial drugs are selected from levofloxacin, the metal coordination ions of the fumarate metal-organic framework are Zr(IV) and Fe(III), and the ligand is fumaric acid. The preparation method of the fumarate metal-organic framework comprises: dissolving a Zr(IV) salt, an Fe(III) salt and fumaric acid in an organic solvent, heating to 110-130°C for reaction, collecting the precipitate, and obtaining the fumarate metal-organic framework; the molar ratio of Zr(IV), Fe(III) and fumaric acid is 1:1:3.0-3.3; the organic solvent is selected from N,N-dimethylformamide (DMF) and N-methylpyrrolidone (NMP); and the mass ratio of the other antibacterial drugs to the fumarate metal-organic framework is 1:8000-20000.

2. The pharmaceutical composition according to claim 1, characterized in that The molar ratio of Zr(IV), Fe(III) and fumaric acid is 1:1:3.0-3.

125.

3. The pharmaceutical composition according to claim 1, characterized in that The average particle size of the fumarate metal organic framework is 100-300 nm.

4. The pharmaceutical composition according to claim 1, characterized in that The average particle size of the fumarate metal organic framework is 150-250 nm.

5. Use of the pharmaceutical composition according to any one of claims 1 to 4 in the preparation of a drug for antibacterial or anti-biofilm use.

6. The use according to claim 5, characterized in that The medicine is used for treating bacterial infection of the eye.

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