Construction method of novel marine source 4-hydroxybenzoxazole-2 (3H)-thioketone carrier based on beta-cyclodextrin metal organic framework and application of novel marine source 4-hydroxybenzoxazole-2 (3H)-thioketone carrier in enhancement of antibacterial activity

Abstract

The invention relates to the field of biological medicine, in particular to a construction method of a novel marine source 4-hydroxybenzoxazole-2 (3H)-thioketone carrier based on a beta-cyclodextrin metal organic framework and application of the novel marine source 4-hydroxybenzoxazole-2 (3H)-thioketone carrier to antibacterial activity enhancement. According to the invention, the effect of 4-hydroxybenzoxazol-2 (3H)-thioketone (HBOT) on resisting escherichia coli and shigella flexneri is found. The result shows that the antibacterial diameter of the HBOT on shigella flexneri is 15.78 + / -0.67 mm, and the antibacterial diameter of the HBOT on escherichia coli is 10.81 + / -0.50 mm; the MIC of the free HBOT to shigella flexneri is 128 [mu] g / mL, and the MIC of the beta-CD-MOF loaded with the free HBOT is 64 [mu] g / mL, so that the beta-CD-MOF loaded with the free HBOT has the capability of enhancing the antibacterial performance.

Description

[0001] The present invention relates to the field of biomedicine, and in particular to a method for constructing a new marine-derived 4-hydroxybenzoxazole-2(3H)-thione carrier based on a β-cyclodextrin metal-organic framework and its application in enhancing antibacterial activity. Background Art

[0002] Escherichia coli (E. coli) is a short, Gram-negative bacillus with peritrichous flagella, motile, and non-spore-forming bacteria. It ferments a variety of sugars to produce acid and gas, and is a normal inhabitant of the human and animal intestines. E. coli can infect the body through contaminated food or water. Infection with E. coli can cause not only diarrhea and food poisoning, but also serious consequences such as renal failure.

[0003] Shigella is one of the main pathogens of gastrointestinal infections. The genus Shigella is divided into four species and at least 47 serotypes, of which Shigella flexneri is the dominant bacterium. Studies have shown that accidentally ingesting food contaminated with Shigella flexneri is the main cause of diarrhea.

[0004] Bacterial contamination is a serious problem in food safety and the food production industry. With the increasing emergence of drug-resistant strains, using antibiotics to prevent bacterial contamination is no longer the optimal approach. Drug-resistant strains can evolve into new resistant strains in the environment, and the development of antibiotics far outstrips the rate at which these strains emerge. Therefore, the search for new drugs against Escherichia coli and Shigella flexneri is urgent.

[0005] Metal-organic frameworks (MOFs) are a new type of porous material with large specific surface area, good pore structure and high stability. However, most MOFs cannot be directly used in drug loading research due to the toxicity of their constituent raw materials. In contrast, cyclodextrin metal-organic frameworks (β-CD-MOFs) have the advantages of safety, non-toxicity, renewability, environmental friendliness and excellent biocompatibility, and have been widely studied as carrier materials. However, the application of β-CD-MOF in drug delivery systems is limited by its relatively low toxicity, and the synthesis process of β-CD-MOF is complex and the yield is not ideal. Therefore, it is of great research value to achieve rapid synthesis of β-CD-MOF and improve its antibacterial efficacy by combining it with marine antibacterial agents. This method can solve the problems of low yield of β-CD-MOF and weak antibacterial ability of HBOT, thereby broadening its application range in drug sustained-release loading. The changes in metal centers and ligands in MOFs are aimed at increasing their specific surface area (up to 10,000 m 2 / g, with a porosity of approximately 50% of the material's volume), far exceeding that of traditional porous materials such as zeolites and activated carbon. The MOF framework is highly functional, allowing for diverse structures and applications through chemical or post-synthetic modification. The choice of metal and its coordination properties further enhance the diversity of MOF structures. The shape and size of MOF pores can be customized to accommodate different drugs, providing flexibility for drug delivery. Due to their non-toxicity and high drug loading capacity, MOFs are widely used in fields such as separation, catalysis, chemical sensing, and drug delivery, demonstrating enormous potential for scientific and industrial applications. Cyclodextrin (CD) is a natural cyclic oligosaccharide composed of D-pyranose glucose units linked by α-1,4-glycosidic bonds. It is classified as α-CD, β-CD, and γ-CD depending on the number of glucose units (6, 7, or 8). CDs are approximately 0.8 nm in height, with diameters of 0.57, 0.78, and 0.95 nm, respectively. Due to the spatial arrangement and linkage of glucose units, CDs have a hollow tubular structure that is wide at the top and narrow at the bottom. With the advent of industrialized cyclodextrin production, its readily available and "hydrophilic on the outside, hydrophobic on the inside" properties have made it extremely valuable in the food and pharmaceutical industries. In particular, its unique cavity structure holds great potential for drug delivery. β-Cyclodextrin metal-organic frameworks (β-CD-MOFs) are porous crystalline materials formed by the coordination of cyclodextrin with alkali metal ions. Traditionally, they are synthesized by vapor diffusion. In recent years, new methods, including improved vapor diffusion, hydrothermal synthesis, ultrasound technology, microwave-assisted methods, and seeded growth techniques, have been developed. However, these methods face challenges such as complex preparation processes and unclear optimal synthesis conditions, limiting their scalable application. Therefore, exploring the optimal synthesis conditions for β-CD-MOFs is crucial for promoting their widespread application. 4-Hydroxybenzoxazole-2(3H)-thione (HBOT) is the electronic isostere of the alkaloid 4-hydroxy-2-benzoxazolone from the mangrove plant Acanthusilicifolius Linnaeus. However, the limited availability and slow growth of Acanthusilicifolius linnaeus have hampered its drug development. Structurally, HBOT consists of a benzene ring and a five-membered ring containing nitrogen and oxygen heteroatoms, making it a class of biologically active compounds. HBOT has low toxicity and exhibits certain antibacterial properties. The anti-inflammatory effects of some of its derivatives are comparable to those of traditional anti-inflammatory drugs such as aspirin. Benzoxazole-2-thione, the core component of HBOT, also exhibits antibacterial, anti-inflammatory, and analgesic effects. The presence of sulfur atoms enhances its lipid solubility, allowing it to easily penetrate biomembranes and act on cells. To expand its application, reducing toxicity and further enhancing antibacterial activity are key. Despite increasing research on cyclodextrin-based MOFs and marine-derived antimicrobial agents, integrating them into a unified drug delivery system remains underexplored. To this end, the present invention proposes to synthesize a β-CD-MOF platform using an ultrasound-assisted method optimized by Box-Behnken response surface methodology, achieving high yield and structural uniformity.This system was used to encapsulate HBOT, a substance with limited solubility but promising antimicrobial potential, into a layered carrier with enhanced porosity and pH-responsive release behavior. To our knowledge, this is the first study to utilize a green-synthesized hybrid system of β-cyclodextrin and MOF for controlled delivery of HBOT. Comprehensive kinetic modeling, cytocompatibility analysis, and antibacterial validation against Gram-negative strains were performed. This approach provides a novel and scalable approach for developing biocompatible and pH-responsive antimicrobial carriers. Summary of the Invention

[0006] The purpose of the present invention is to provide a method for constructing a new marine-derived 4-hydroxybenzoxazole-2(3H)-thione carrier based on a β-cyclodextrin metal-organic framework and its application in enhancing antibacterial activity, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides application of 4-hydroxybenzoxazole-2(3H)-thione in preparing medicines for resisting Escherichia coli and Shigella flexneri.

[0009] The invention provides a drug delivery system for resisting Escherichia coli and Shigella flexneri. The drug delivery system comprises a carrier loaded with 4-hydroxybenzoxazole-2(3H)-thione; the carrier is a β-cyclodextrin metal organic framework.

[0010] Preferably, the preparation method of the β-cyclodextrin metal-organic framework comprises the following steps:

[0011] β-cyclodextrin, potassium hydroxide and water are mixed evenly, and then mixed with methanol and placed in a water bath to obtain a mixed solution;

[0012] The mixed solution is mixed evenly with polyethylene glycol, and then subjected to ultrasonic treatment, crystallization treatment, centrifugation treatment and drying in sequence to obtain the β-cyclodextrin metal-organic framework.

[0013] Preferably, the molar ratio of β-cyclodextrin to potassium hydroxide is 1:8.

[0014] Preferably, the water bath time is 10 min and the temperature is 60°C;

[0015] The ultrasonic treatment time is 10 minutes and the temperature is 60°C;

[0016] The crystallization treatment time is 24h;

[0017] The centrifugal treatment time is 5 min and the rotation speed is 5000 rpm;

[0018] The drying time is 24 hours and the temperature is 60°C.

[0019] The present invention provides a method for preparing the above-mentioned drug delivery system, which comprises the following steps:

[0020] β-cyclodextrin, potassium hydroxide and water are mixed evenly, and then mixed with methanol and placed in a water bath to obtain a mixed solution;

[0021] The mixed solution is uniformly mixed with polyethylene glycol, and sequentially subjected to ultrasonic treatment, crystallization treatment, centrifugation treatment and drying to obtain the β-cyclodextrin metal-organic framework;

[0022] Mixing 4-hydroxybenzoxazole-2(3H)-thione and acetonitrile to obtain HBOT-acetonitrile solution;

[0023] The β-cyclodextrin metal organic framework and the HBOT-acetonitrile solution are mixed evenly to obtain the drug delivery system.

[0024] Preferably, the molar ratio of β-cyclodextrin to potassium hydroxide is 1:8.

[0025] Preferably, the water bath time is 10 min and the temperature is 60°C;

[0026] The ultrasonic treatment time is 10 minutes and the temperature is 60°C;

[0027] The crystallization treatment time is 24h;

[0028] The centrifugal treatment time is 5 min and the rotation speed is 5000 rpm;

[0029] The drying time is 24 hours and the temperature is 60°C;

[0030] The mass volume ratio of the β-cyclodextrin metal organic framework and the HBOT-acetonitrile solution is 5 mg:1 mL.

[0031] The present invention provides use of the above-mentioned drug delivery system in the preparation of an antibacterial agent, wherein the antibacterial agent targets bacteria including Escherichia coli and / or Shigella flexneri.

[0032] The present invention provides an antimicrobial agent, comprising the above-mentioned drug delivery system; the bacteria targeted by the antimicrobial agent include Escherichia coli and / or Shigella flexneri.

[0033] The present invention discloses the following technical effects:

[0034] The present invention discovered the antibacterial effect of 4-hydroxybenzoxazole-2(3H)-thione on Escherichia coli and Shigella flexneri. In a specific embodiment of the present invention, the inhibition zone diameter and MIC of 4-hydroxybenzoxazole-2(3H)-thione were evaluated by the Oxford cup method. The results showed that the inhibition zone diameter of 4-hydroxybenzoxazole-2(3H)-thione against Shigella flexneri was 15.78±0.67 mm, and the inhibition zone diameter against Escherichia coli was 10.81±0.50 mm; the MIC of free 4-hydroxybenzoxazole-2(3H)-thione against Shigella flexneri was 128 μg / mL, and the MIC of β-CD-MOF loaded with free 4-hydroxybenzoxazole-2(3H)-thione was 64 μg / mL. It can be seen that β-CD-MOF loaded with free 4-hydroxybenzoxazole-2(3H)-thione has the ability to enhance antibacterial performance.

[0035] 4-Hydroxybenzoxazole-2(3H)-thione (HBOT) is an isostere of the alkaloid 4-hydroxy-2-benzoxazolone from the mangrove plant Acanthusilicifolius Linnaeus. However, due to its limited source and slow growth, research and development of its biological activity is restricted. To reduce the toxicity of 4-Hydroxybenzoxazole-2(3H)-thione and further enhance its antibacterial effect, the present invention provides a drug delivery system (HBOT / β-CD-MOF or HBOT / β-CD-MOF complex), which includes a carrier loaded with 4-Hydroxybenzoxazole-2(3H)-thione, wherein the carrier is a β-cyclodextrin metal-organic framework. The present invention integrates β-cyclodextrin (β-CD) into a metal-organic framework (MOF) structure (i.e., β-CD-MOF), resulting in a highly efficient and biocompatible carrier with enhanced physicochemical properties suitable for pH-responsive drug delivery. The HBOT / β-CD-MOF complex exhibited excellent antibacterial properties against Shigella flexneri and Escherichia coli, with an inhibition zone of 21.54±0.4 mm, significantly outperforming free HBOT through sustained drug release. CCK-8 cytotoxicity assays confirmed its high biocompatibility, with a cell survival rate exceeding 103% at a concentration of 800 μg / mL.

[0036] In a specific embodiment of the present invention, β-CD-MOF was synthesized by ultrasound-assisted method and optimized by Box-Behnken response surface method to maximize the yield and structural performance. The results showed that the solvent volume and reaction temperature were key variables. When the molar ratio was 8:1 (KOH:β-CD), the solvent was 20mL, and the temperature was 60°C, the optimal yield reached 82.39%. Subsequently, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and nitrogen adsorption-desorption isotherm characterization confirmed that β-CD-MOF was successfully formed with a high surface area (332.13m 2 / g) layered structure, and the surface area was slightly reduced to 247.07 m after encapsulating 4-hydroxybenzoxazole-23H)-thione (HBOT). 2 / g; In vitro release studies showed that it has pH-dependent release kinetics: at pH 2, it is mainly based on first-order diffusion (R 2 =0.977), while at pH 7, due to the combined effects of swelling and diffusion mechanisms, the non-Fickian diffusion described by the Ritger-Peppas equation (R 2 =0.97, n=0.70); the HBOT / β-CD-MOF composite exhibited excellent antibacterial properties against Shigella flexneri and Escherichia coli; significantly outperforming free HBOT through sustained drug release; and CCK-8 cytotoxicity assays confirmed its high biocompatibility, with cell viability exceeding 103% at a concentration of 800 μg / mL. Overall, this invention provides a scalable and environmentally friendly approach for developing multifunctional MOF-based carriers with responsive release properties, offering promising potential for safer, long-lasting, and targeted antibacterial therapies. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0038] Figure 1 The preparation process of HBOT / β-CD-MOF;

[0039] Figure 2 Response surface diagram and contour diagram of the interaction between raw material molar ratio and methanol dosage (A), raw material molar ratio and ultrasonic temperature (B), and methanol dosage and ultrasonic temperature (C) on the yield of β-CD-MOF;

[0040] Figure 3Scanning electron microscopy images of β-CD-MOF (A and C) and HBOT / β-CD-MOF (B and D);

[0041] Figure 4 Fourier transform infrared spectra of KOH, β-CD, β-CD-mof, HBOT and HBOT / β-CD-mof (A) and X-ray diffraction patterns of KOH, β-CD-MOF and HBOT / β-CD-MOF (B);

[0042] Figure 5 Performance evaluation of HBOT / β-CD-MOF and β-CD-MOF; A is the comparison of the swelling behavior of HBOT / β-CD-MOF and β-CD-MOF in neutral buffer (25°C, pH 7.0); B is the adsorption kinetics curve at different HBOT concentrations; C is the equilibrium isotherm at different HBOT concentrations; DI is the pseudo-first-order, pseudo-second-order, Elovich, Scatchard, Langmuir, and Freundlich model analysis of the adsorption mechanism; J is the N2 adsorption-desorption isotherm of porosity analysis; K and L are the in vitro release profiles in artificial gastric fluid (AGF, pH 2.0) and artificial intestinal fluid (AIF, pH 7.0), respectively, showing pH-dependent sustained release;

[0043] Figure 6 To detect AGF (1h, a; 3h, c; 6h, e) and AIF (1h, b; 9h, d; 20h, f) of HBOT-β-MOF at different times by HPLC;

[0044] Figure 7 The effects of HBOT, β-CD-MOF, and HBOT / β-CD-MOF on MODE-K cell activity; A is a control without sample; B is 800 μg / mL β-CD-MOF; C is 800 μg / mL HBOT / β-CD-MOF; D is the effect of different concentrations of different substances on cell survival rate;

[0045] Figure 8Evaluation of the antibacterial activity of HBOT / β-CD-MOF against Gram-negative pathogens; A is the evaluation of the antibacterial activity of HBOT / β-CD-MOF against Shigella flexneri, 1 is the blank control (0 μg / mL), 2 is β-CD-MOF, 3 is HBOT / β-CD-MOF, 4 is free HBOT, and 5 is the positive control (vancomycin 0.01 μg / mL); B is the evaluation of the antibacterial activity of HBOT / β-CD-MOF against Escherichia coli, 1 is the blank control (0 μg / mL), 2 is β-CD-MOF, 3 is HBOT / β-CD-MOF, 4 is free HBOT, and 5 is the positive control (vancomycin 0.01 μg / mL); C is the quantitative analysis of inhibition diameter (mean ± SEM, n = 3), compared with the blank control, *p < 0.05, **p < 0.01; #p < 0.05, ##p < 0.01; D is the statistical results of the inhibition rate of HBOT / β-CD-MOF against Salmonella flexneri (n = 3; #p < 0.05, ##p < 0.01 vs free HBOT). DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0047] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0048] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0049] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0050] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0051] Example 1 Preparation of HBOT / β-CD-MOF

[0052] (1) Experimental methods

[0053] 1. Chemical reagents

[0054] 4-Hydroxybenzoxazole-2(3H)-thione (HBOT, purity ≥98%, verified by HPLC) was purchased from the China Food and Drug Administration (Beijing). β-Cyclodextrin (β-CD, purity ≥98.0%) and potassium hydroxide (KOH, purity ≥99.0%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd. Polyethylene glycol-8000 (PEG-8000, molecular weight: 7000-9000, purity >99%) was purchased from Aladdin Reagent (Shanghai) Co., Ltd. Methanol (analytical grade, purity >98%) was purchased from Beijing Chemical Reagent Factory.

[0055] 2. Instruments and equipment

[0056] The framework materials were characterized by Fourier transform infrared spectroscopy (FT-IR; PU9800, PANalytical, Almelo, The Netherlands). The surface morphology was observed by scanning electron microscopy (SEM, GeminiSEM-300 microscope, Zeiss, Oberkochen, Germany) with an instrument operating voltage of 3 kV to capture clear and complete surface images. HBOT quantitative analysis was performed using a Waters E2695+UV high-performance liquid chromatograph. The products were synthesized using a CNC variable temperature ultrasonic cleaner (KQ250DE, 4 kHz) from Shanghai Jingqi Instrument Co., Ltd. for ultrasonic and thermal treatment of the reaction. The adsorption and release characteristics of HBOT / β-CD-MOF were analyzed by high-performance liquid chromatography (HPLC, Agilent Infinity II 1260 system, Agilent, Waldbronn, Germany) equipped with a PAD detector (set wavelength of 292 nm). Separation was performed using a C-ion column chromatography system from Nacalai Tesque (Kyoto, Japan). 18 Chromatographic column (COSMOSILC 18 The column was 2.1 mm × 150 mm, 5 μm, and the temperature was 25°C. The mobile phase consisted of acetonitrile (A) and ultrapure water (B), with an injection volume of 20 μL and a flow rate of 1 mL / min. Data analysis and graphics were performed using Design-Expert 13.0 software (Stat-Ease, USA). The Escherichia coli model was ATCC 25922, and the Shigella flexneri model was ATCC 12022, both gifts from the Lanzhou Institute of Animal Husbandry and Veterinary Medicine, Chinese Academy of Agricultural Sciences.

[0057] 3. Ultrasound-thermal assisted preparation of HBOT-loaded cyclodextrin metal-organic frameworks

[0058] Preparation process as Figure 1 As shown. β-CD, KOH and 20 mL of deionized water were added to a flask, stirred at room temperature until completely dissolved, and then filtered through a 0.45 μm filter membrane. In addition, 20 mL of methanol was added to a 100 mL round-bottom flask, and the filtrate was added to form a milky white solution. The mixture was placed in a 60°C water bath and heated for 10 minutes until a clear solution was obtained. The solution was then transferred to an ultrasonic cleaner (4000 Hz) at different temperatures. Polyethylene glycol-8000 (PEG-8000, 108 mg) was added before ultrasonic treatment, and the solution was ultrasonically treated for 10 minutes. Afterwards, the solution was placed at room temperature and transferred to a 50 mL centrifuge tube for crystallization for 24 hours. The crystallized solution was centrifuged at 5000 rpm for 5 minutes, and the resulting product was dried in a constant temperature oven at 60°C for 24 hours. After cooling to room temperature, β-cyclodextrin metal organic framework (β-CD-MOF) material was obtained by ultrasound-assisted preparation. Subsequently, HBOT was loaded onto β-CD-MOF through different concentration gradients to prepare HBOT / β-CD-MOF composites (see step “dynamic and static adsorption tests” for details).

[0059] 3. Single-factor experiment

[0060] The control variable method was used to keep other factors constant and evaluate the impact of a single variable on the experimental results. Factors affecting the yield of β-CD-MOF (including the molar ratio of KOH to β-CD, methanol dosage, and ultrasonic temperature) were selected for single-factor experiments to evaluate the impact of different levels of each factor on the yield of β-CD-MOF. The three factors with the most significant impact were then selected for the Box-Behnken response surface experiment. Since the theoretical mass of the CD-MOF material cannot be calculated, the mass of β-CD was used instead of the theoretical mass, and the following yield formula was obtained:

[0061] Yield (%) = m1 / m2 × 100%;

[0062] Where m1 is the mass of β-CD-MOF and m2 is the mass of β-CD.

[0063] 5. Box-Behnken response surface experiment

[0064] Based on the results of single-factor experiments, three key influencing factors were identified: molar ratio A, solvent volume B, and reaction temperature C. Based on the Box-Behnken response surface design principle, a three-factor, three-level experimental model was established with β-CD-MOF yield as the response variable. The molar ratio (A) was 6:1, 8:1, and 10:1 (corresponding to -1, 0, and +1 levels), the solvent volume (B) was 15, 20, and 25 mL (corresponding to -1, 0, and +1 levels), and the reaction temperature (C) was 50, 60, and 70°C (corresponding to -1, 0, and +1 levels). Response surface optimization experiments were then conducted.

[0065] 6. Swelling ratio experiment

[0066] Dried samples of HBOT / β-CD-MOF and β-CD-MOF were immersed in 0.9% NaCl solution at 37°C. Samples were removed every 10 minutes, the surface water was gently removed, and the samples were weighed. This process lasted for 120 minutes. The swelling ratio was calculated at each time point to evaluate the swelling behavior. The swelling kinetics analyzed the rapid swelling of β-CD-MOF, which is attributed to its high permeability, and the stable saturation swelling curve of HBOT / β-CD-MOF. The swelling ratio calculation formula is as follows:

[0067] SR=(W t -W0) / m;

[0068] Where W t is the mass of the polymer at time t after swelling, W0 is the mass of the initial polymer, and m is the mass of the polymer after weighing.

[0069] 7. Dynamic and static adsorption tests

[0070] Two centrifuge tubes were each loaded with 5 mg of β-CD-MOF. One tube was injected with 1 mL of HBOT-acetonitrile solution (HBOT-acetonitrile solution was prepared by mixing different masses of 4-hydroxybenzoxazole-2(3H)-thione with acetonitrile) at different concentrations (10, 30, 50, 100, 250, 500, 800, and 1000 μg / mL), while the other tube was injected with 1 mL of 1000 μg / mL HBOT-acetonitrile solution. After thorough mixing, the tubes were allowed to stand for predetermined time intervals (60, 120, 180, 240, 300, and 360 min) and then centrifuged at 5000 rpm for 5 min. The supernatant was collected, filtered through a 0.22 μm membrane, and analyzed by HPLC. The adsorption amount (Q (μg / mg)) was determined by the following formula:

[0071] Q = (C0 - Ce) / m × V;

[0072] Where C0 is the initial concentration, Ce is the concentration of the solution after adsorption, V is the volume of the solution, and m is the mass of β-CD-MOF.

[0073] In this experiment, the following adsorption models were used to fit the data: pseudo-first-order kinetic model, pseudo-second-order kinetic model, Elovich model, Scatchard model, Langmuir model, and Freundlich model. The specific formulas used are as follows:

[0074] Pseudo-first-order kinetic model:

[0075] Q t =Q e (1-e -kt );

[0076] Pseudo-second-order kinetic model:

[0077]

[0078] Where Q e is the adsorption amount at adsorption equilibrium, Q t is the adsorption amount at time t, and k is the pseudo-first-order adsorption kinetic parameter.

[0079] Elovich Model:

[0080]

[0081] Where α is the initial adsorption rate, β is the adsorption constant, and Q t is the adsorption amount at time t.

[0082] Scatchard model:

[0083]

[0084] Where Q max is the maximum saturated adsorption capacity, and Ks is the dissociation constant.

[0085] Langmuir model:

[0086]

[0087] Where C e is the concentration of the solution after adsorption, A and K are constants.

[0088] Freundlich model:

[0089]

[0090] Where C e is the concentration of the solution after adsorption, K is a constant, and n is the adsorption index.

[0091] 8. Nitrogen adsorption experiment

[0092] β-CD-MOF and HBOT / β-CD-MOF were pretreated to remove surface impurities and moisture. Subsequently, the samples were exposed to nitrogen at liquid nitrogen temperature (approximately 77K) and the adsorption capacity at different pressures was measured.

[0093] 9. In vitro sustained-release experiment

[0094] Artificial gastric fluid (AGF, pH = 2) and artificial intestinal fluid (AIF, pH = 7) were prepared according to the method described in the literature "Development of an Fe3O4 Surface-Grafted Carboxymethyl Chitosan Molecularly Imprinted Polymer for Specific Recognition and Sustained Release of Salidroside". 50 mg of pre-prepared HBOT / β-CD-MOF was added to 10 mL of AGF or AIF and thoroughly shaken in a constant temperature water bath shaker at 37°C and 180 rpm. The reaction in AGF lasted for 6 hours, and 1 mL of supernatant was collected by centrifugation every hour, and then 1 mL of fresh AGF was added to the system. In contrast, the reaction in AIF was extended to 36 hours, and 1 mL of samples were taken at 1, 3, 6, 9, 18, and 24 hours, respectively. After each sampling, 1 mL of fresh AIF was added to the reaction system. The cumulative release rate (DS%) of HBOT was calculated as follows:

[0095]

[0096] Where V t represents the volume of the released medium at each sampling, n represents the number of sampling times, C i represents the concentration of HBOT in the release medium at the i-th moment, V o represents the total volume of the release medium, C n represents the concentration of release medium, M ∞ The experiment was performed three times, and the results were averaged for analysis.

[0097] 10. In vitro drug release kinetics

[0098] The present invention uses multiple kinetic models to analyze the drug release process of HBOT / β-CD-MOF. The relevant model equations are shown below:

[0099] D t =Kt+D0;

[0100] Where D t is the cumulative drug release rate at time t, K is the zero-order release constant, D0 is the initial drug concentration, and t is the release time.

[0101] D t =A(1-e -kt );

[0102] Where D t is the cumulative release rate of the drug at time t, k is the first-order release constant, D ∞ is the maximum cumulative release percentage, and A is a constant.

[0103]

[0104] Where D t is the amount of drug released at time t, K h is the release rate constant.

[0105] D t =K p t n ;

[0106] Where D t is the cumulative release rate of the drug at time t, K p is the kinetic constant, and n is the release exponent.

[0107]

[0108] Where D t It represents the ratio of the released amount at time t to the total adsorption amount, k is the release rate constant, and n is the release index.

[0109] 11. Cytotoxicity test

[0110] Mouse intestinal epithelial cells were plated at 1×10 3 Cells were seeded at a density of 100 cells in a 96-well plate and treated with different concentrations of HBOT (50, 100, 200, 400 and 800 μg / mL) or HBOT / β-CD-MOF complex (corresponding concentrations were 178.63, 357.27, 714.54, 1429.08 and 2858.16 μg / mL, respectively, with a loading rate of 27.99%). A β-CD-MOF control group receiving an equivalent carrier dose was also set up. After incubation for 24 hours, 10 μL of CCK-8 solution was added to each well and incubated for another 2 hours at 37°C and 5% CO2. The absorbance was then measured at 490 nm using a microplate reader, and the cell viability was calculated to assess cytotoxicity. Note: Only the final concentration of HBOT is marked in the experimental data, and the same marking method was used for the antibacterial experiment. The formula for calculating cell viability is as follows:

[0111]

[0112] Where, is the average value of the normal control group, G n are the measured values for each group.

[0113] 12. Determination of the minimum inhibitory concentration (MIC) of compounds against bacteria

[0114] The diameter of the inhibition zone was assessed using the Oxford cup method. Specifically, 0.2 mL of 64 μg / mL HBOT, 0.2 mL of 228.66 μg / mL HBOT / β-CD-MOF, 0.2 mL of 228.66 μg / mL β-CD-MOF, and 0.2 mL of 0.1 μg / mL vancomycin (positive control) were added to separate Oxford cups. The plates were incubated at 37°C under controlled temperature and humidity for 24 hours. After incubation, the inhibition zone was visually inspected and measured.

[0115] The antibacterial activity of HBOT, β-CD-MOF and HBOT / β-CD-MOF complex against Escherichia coli and Shigella flexneri was quantitatively tested by the microbroth double dilution method. A bacterial suspension with a turbidity equivalent to 0.5 McFarland standard was prepared and inoculated into a 96-well plate. 0.1% vancomycin was used as a positive control (PC) and untreated blank wells were used as negative controls (BC). HBOT was serially diluted from 128 μg / mL to 1 μg / mL, while the HBOT / β-CD-MOF complex was diluted from 457.31 μg / mL to 3.57 μg / mL. The plates were incubated at 37°C for 24 h. The concentrations of HBOT and β-CD-MOF were determined by spectrophotometry at 600 nm (OD 600 ) to monitor bacterial growth and determine the minimum inhibitory concentration (MIC). The antibacterial effect was calculated using the following formula:

[0116]

[0117] Where ODc is the OD value of the blank control group, and ODe is the OD value of the experimental group.

[0118] (2) Results

[0119] 1. Optimization of single-factor synthesis conditions of β-CD-MOF

[0120] Porous MOF materials have been used for controlled drug release to achieve sustained and efficient delivery of therapeutic agents, thus broadening the functional scope of MOFs in biomedical applications. Here, the Box-Behnken response surface methodology was used to optimize the synthesis parameters. Statistical analysis of the model showed that the solvent volume (factor B) and its quadratic term B 2 and C 2The yield of β-CD-MOF was significantly affected (P<0.001). Under the optimized conditions of A=8:1 (KOH:β-CD) molar ratio, B=20 mL solvent volume and C=60°C reaction temperature, the experimental yield reached 82.39%. Figure 2 shown.

[0121] 2. Optimization of synthesis conditions based on Box-Behnken response surface methodology

[0122] The three main factors affecting β-CD-MOF are the raw material molar ratio, methanol volume fraction, and ultrasonic temperature. Taking the β-CD-MOF yield as the response value, the multivariate quadratic regression equation of the yield was obtained by fitting the experimental data as shown below:

[0123] Y=-466.914+7.3273A+15.587B+11.5003C+0.647AB-0.029AC+0.1409BC-1.2416A 2 -0.6639B 2 -0.1186C 2 .

[0124] The β-CD-MOF or HBOT / β-CD-MOF used in subsequent experiments were prepared using the optimized conditions obtained here (A = 8:1 (KOH:β-CD) molar ratio, B = 20 mL solvent volume, and C = 60°C reaction temperature, with an experimental yield of 82.39%); at the same time, the concentration of HBOT in the HBOT-acetonitrile solution used to prepare HBOT / β-CD-MOF was 1000 μg / mL.

[0125] 3. Scanning electron microscopy (SEM) analysis

[0126] SEM analysis showed that the microstructure of β-CD-MOF after loading HBOT changed from a dense morphology to a relatively loose layered structure ( Figure 3 A and C in Figure ). The intrinsic sheet structure of β-CD-MOF ( Figure 3 B and D) and β-CD and K + The lack of strong covalent bonds between them may cause columnar displacement after HBOT encapsulation, resulting in loose arrangement. Due to the high polarity of HBOT, its ability to be directly adsorbed by β-CD is limited, suggesting that the observed layered structure involves not only physical aggregation but also the structural transformation of the MOF itself. + The ionic interactions between the β-CD-MOF and the β-CD-MOF may promote the formation of this layered framework. This unique structure enables the efficient encapsulation of HBOT in micropores of 1-5 μm, providing a larger specific surface area and stronger binding interactions than unmodified β-CD-MOF, thereby effectively retaining HBOT within the porous matrix.

[0127] 4. Fourier transform infrared spectroscopy (FTIR) analysis

[0128] The infrared spectra of KOH, β-CD, β-CD-MOF, HBOT and HBOT / β-CD-MOF are as follows: Figure 4 The characteristic absorption band of KOH is 3583 cm -1 (-OH stretching vibration). The characteristic peak of β-CD is located at 3591.9 cm -1 、1623.2cm -1 and 1137.2cm -1 There are four obvious characteristic absorption bands in β-CD-MOF, among which 2931m -1 The C-H stretching vibration of -CH3 and -CH2 is 1612.2 cm -1 and 1147.3cm -1 The peak at 3611 cm indicates CC, CO or CCO stretching vibration. There is no significant difference in the infrared spectra of β-CD-MOF and β-CD, indicating the presence of β-CD skeleton in the synthetic material. -1 The blue shift of CO and OH peaks at 1616 cm-1 is attributed to the formation of KO and Na-O bonds, confirming the successful synthesis of β-CD-MOF. -1 and 1504cm -1 The peak at 1265 cm is attributed to the stretching vibration of the benzene ring skeleton, and the CH stretching vibration peak is located at 1265 cm -1 , -C=S stretching vibration peak is located at 1002cm -1 The hydroxyl absorption peak of HBOT / β-CD-MOF red-shifted to 3452 cm -1 And the frequency increased, suggesting that there was an interaction between the OH of β-CD-MOF and HBOT, indicating that HBOT / β-CD-MOF was successfully synthesized.

[0129] 5. X-ray diffraction (XRD) analysis

[0130] To further study the intermolecular interactions, the XRD patterns of KOH, β-CD-MOF and HBOT / β-CD-MOF were shown in Figure 2. Figure 4 As shown in Figure B, 12-30° is the characteristic diffraction peak of KOH and β-CD-MOF. The spectrum of β-CD-MOF shows that multiple diffraction peaks are slightly shifted, disappeared or replaced by clearer characteristic peaks, indicating that β-CD and KOH react during the synthesis process. + The interaction formed a more ordered crystal structure. In addition, the loading of HBOT into β-CD-MOF did not change the crystalline state of β-cyclodextrin.

[0131] 6. High Performance Liquid Chromatography (HPLC) Method Validation

[0132] According to IUPAC recommendations and previous reports, the calibration curve of HBOT is good in the range of 1-1000 μg / g. The regression equation of HBOT standard solution is y=0.9808x+6.7714, and the correlation coefficient (R 2 ) was 0.9996, and the limits of detection and quantification were 0.22-2.0 μg / g and 2.50 μg / g, respectively. The intra-day precision of HBOT at the same concentration during adsorption and release was determined five times, and the calculated RSD was 1.26%-2.15%.

[0133] 7. Swelling properties

[0134] like Figure 5 As shown in Figure A, the swelling rates of HBOT / β-CD-MOF and β-CD-MOF change with time, initially increasing and then decreasing after 100 minutes. Notably, the swelling behavior of HBOT / β-CD-MOF gradually stabilizes after reaching a more pronounced saturation state. In contrast, β-CD-MOF cross-linked with HBOT molecules exhibits a higher swelling rate due to its higher permeability and solvent uptake capacity. These results indicate that β-CD-MOF possesses a larger adsorption area and drug-loading capacity.

[0135] 8. Static and dynamic adsorption

[0136] The adsorption performance of β-CD-MOF under dynamic and static conditions is shown in Figure 2. Figure 5 As shown in Figures B and C. During the dynamic process, the adsorption effect was significant within the initial 300 min. During static adsorption, β-CD-MOF reached saturation at a concentration of 800 μg / mL, with a drug loading rate of 27.99 ± 3.12%. The adsorption kinetics and isotherms, dynamic fitting of pseudo-first-order and pseudo-second-order equations, and Elovich plots are shown in Figures B and C. Figure 5 The static fitting of Scatchard, Langmuir and Freundlich equations is shown in Figure 5 The corresponding parameters are shown in Table 1. The pseudo-first-order model is suitable for adsorption behavior when the adsorbent concentration is high, the pseudo-second-order model is used to characterize the physical and chemical adsorption process, and the Elovich model is often used to describe the adsorption of liquid-solid systems. The dynamic adsorption data is more consistent with the pseudo-first-order model (R 2>0.99). This model is applicable to saturated homogeneous adsorbents (such as molecularly imprinted polymers), while the pseudo-secondary model assumes that adsorption occurs on heterogeneous surfaces and that interactions exist between adsorbed molecules. The Scatchard model plays an important role in the theory of regular and non-electrolyte solutions. The Langmuir equation assumes that adsorption on heterogeneous surfaces occurs through intermolecular interactions and is more applicable to static adsorption conditions. Results indicate that the adsorption of HBOT on β-CD-MOF involves a monolayer adsorption process.

[0137] Table 1 Isotherm and kinetic model correction equations and correlation coefficients of HBOT / β-CD-MOF

[0138]

[0139]

[0140] 9. Nitrogen adsorption-desorption isotherm analysis

[0141] The nitrogen adsorption-desorption isotherm curves of HBOT / β-CD-MOF and β-CD-MOF were evaluated by the pressure pump method, as shown in Figure 5 As shown in J in Figure 2. Both materials form stacked pores in the sheet structure. Compared with β-CD-MOF, the specific surface area and pore volume of HBOT / β-CD-MOF gradually increase, and the hysteresis loop improves the mesoporous regularity. However, after loading HBOT, the pore size gradually decreases and the crystallinity increases. The Brunauer-Emmett-Teller (BET) values of HBOT / β-CD-MOF and β-CD-MOF are 247.0650 m 2 / g and 332.1285m 2 / g, indicating that β-CD-MOF is sufficient to adsorb HBOT.

[0142] 10. In vitro sustained release ability

[0143] like Figure 5 As shown in Figure 1, the in vitro sustained release ability of HBOT / β-CD-MOF in artificial gastric fluid (AGF) and artificial intestinal fluid (AIF) was studied. In AGF, the maximum drug release time of HBOT / β-CD-MOF was 250 minutes, and then gradually decreased. The slow rate of decrease indicated that the drug decomposition occurred. For AIF, the cumulative drug release increased rapidly within 10 hours, with a fast release rate in the first 1-10 hours and then gradually slowed down, showing a good sustained release effect ( Figure 5 The release concentration of HBOT / β-CD-MOF in AIF and AGF at different time intervals was determined by HPLC ( Figure 6), and the results showed that its drug release performance in AIF was significantly better than that in AGF. It is speculated that the low pH acidic gastric juice may destroy the HBOT / β-CD-MOF structure, while the weak alkaline intestinal fluid is more conducive to slow drug release.

[0144] 11. Controlled release mechanism

[0145] The release kinetics and potential mechanisms of HBOT from HBOT / β-CD-MOF under different pH conditions were evaluated using various mathematical models, including zero-order, first-order, Higuchi, Ritger-Peppas, Redlich-Peterson, and Weibull (Table 2). The results showed that pH significantly affected the drug release behavior: under acidic conditions (pH = 2), the release curve was best fitted by the first-order kinetic model (R 2 =0.977), indicating that drug diffusion is mainly driven by concentration gradient; under neutral conditions (pH = 7), the Ritger-Peppas model (R 2 =0.974; release index n = 0.7018) showed a non-Fickian diffusion mechanism, which may be caused by the combined effect of carrier swelling and diffusion. The Higuchi model had a poor fit under both pH conditions (R 2 <0.83, suggesting that simple diffusion is not the main release mechanism. In addition, the Weibull model has a strong correlation at pH = 7 (R 2 =0.952), supporting the existence of a more complex pH-responsive release pathway, which may be related to the structural transformation of the MOF matrix. These findings reveal the multi-mechanistic characteristics of drug release and provide a theoretical basis for the design of pH-sensitive delivery systems with tunable release profiles.

[0146] Table 2. Drug release kinetic model parameters.

[0147]

[0148]

[0149] 12. Impact on cell activity

[0150] The results of cell growth and viability assays were as follows: Figure 7 As shown. The intestinal epithelial cells of mice in the blank control group were single-layer, non-overlapping spindle-shaped ( Figure 7 A in the figure); cells in the 800 μg / mL HBOT-treated group showed fragmentation and shrinkage ( Figure 7 B in the figure), while the above phenomenon was significantly alleviated after HBOT / β-CD-MOF pretreatment ( Figure 7 C); β-CD-MOF group had no effect on cell growth ( Figure 7The average cell survival rate of the HBOT / β-CD-MOF group (0-800 μg / mL) was greater than 103.42±3.05%, while the cell growth rate of the 800 μg / mL HBOT group was lower (only 72.76±3.46%). At this concentration, the cell growth of the HBOT / β-CD-MOF group was significantly better than that of the HBOT group (P<0.01), which once again confirmed its safety as a drug carrier ( Figure 7 E in Figure 1). The results also demonstrate that HBOT / β-CD-MOF possesses a protective effect on cells, ultimately resulting in an average cell survival rate exceeding 100%. Interestingly, while HBOT can still stimulate cell growth at concentrations below 400 μg / mL, it exhibits significant cytotoxicity above 400 μg / mL. The sustained-release properties of HBOT / β-CD-MOF effectively enhance the antibacterial safety of HBOT by maintaining an optimal drug concentration in the cell environment.

[0151] 13. Evaluation of antibacterial effect

[0152] like Figure 8 As shown in Figures A and B, β-CD-MOF has no antibacterial effect on Shigella flexneri and Escherichia coli. The antibacterial diameter of HBOT against Shigella flexneri is 15.78±0.67mm, and the antibacterial diameter against Escherichia coli is 10.81±0.50mm. The antibacterial effect of HBOT / β-CD-MOF is significantly better than that of free HBOT (P<0.01), with the antibacterial diameter against Shigella flexneri being 21.54±0.43mm and the antibacterial diameter against Escherichia coli being 18.45±0.46mm, and the antibacterial effect on the former is stronger (P<0.01, Figure 8 C in ). Further research showed that ( Figure 8 D), the antibacterial activity (MIC) of HBOT against Shigella flexneri was 128 μg / mL, and the antibacterial activity of HBOT / β-CD-MOF was 64 μg / mL. Its antibacterial activity against Shigella flexneri was better than that of HBOT (P≤0.05), which may be attributed to its sustained-release effect prolonging the action time of HBOT on bacteria, indicating that HBOT / β-CD-MOF can be used as a new antibacterial agent.

[0153] (3) Conclusion

[0154] This study successfully synthesized a β-cyclodextrin metal-organic framework (β-CD-MOF) via an ultrasound-assisted method. The conditions were optimized using the Box-Behnken response surface methodology, achieving a yield of 82.39% under ideal parameters. This composite material effectively encapsulates the marine-derived compound 4-hydroxybenzoxazole-2(3H)-thione (HBOT), forming a layered structure with excellent porosity and specific surface area, achieving high drug loading efficiency. Compared with traditional cyclodextrin or MOF-based carriers, this hybrid system exhibits enhanced pH-responsive release behavior, enabling sustained and controlled drug delivery under intestinal conditions. Importantly, HBOT / β-CD-MOF exhibited superior antibacterial activity against Shigella flexneri and Escherichia coli and excellent biocompatibility. The combination of a safe material, a scalable green synthesis process, and environmental stability highlights the potential of this system for future applications in biomedicine and pharmaceuticals. Compared with previous studies on MOF or cyclodextrin systems alone, this invention constructs a synergistic carrier with drug stability, release kinetics and biological activity through structural integration, and detailed kinetic modeling and adsorption analysis provide new insights into the drug-carrier interaction mechanism in a variable pH environment.

[0155] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Application of 4-hydroxybenzoxazole-2(3H)-thione in the preparation of drugs against Escherichia coli and Shigella flexneri.

2. A drug delivery system for Escherichia coli and Shigella flexneri, characterized in that: The drug delivery system comprises a carrier loaded with 4-hydroxybenzoxazole-2(3H)-thione; the carrier is a beta-cyclodextrin metal organic framework.

3. The drug delivery system according to claim 2, characterized in that The preparation method of the β-cyclodextrin metal organic framework comprises the following steps: β-cyclodextrin, potassium hydroxide and water are mixed evenly, and then mixed with methanol and placed in a water bath to obtain a mixed solution; The mixed solution is mixed evenly with polyethylene glycol, and then subjected to ultrasonic treatment, crystallization treatment, centrifugation treatment and drying in sequence to obtain the β-cyclodextrin metal-organic framework.

4. The drug delivery system according to claim 3, characterized in that The molar ratio of the β-cyclodextrin to potassium hydroxide is 1:

8.

5. The drug delivery system according to claim 3, characterized in that The water bath time is 10 min and the temperature is 60°C; The ultrasonic treatment time is 10 minutes and the temperature is 60°C; The crystallization treatment time is 24h; The centrifugal treatment time is 5 min and the rotation speed is 5000 rpm; The drying time is 24 hours and the temperature is 60°C.

6. The method for preparing the drug delivery system according to claim 2, wherein: The preparation method comprises the following steps: β-cyclodextrin, potassium hydroxide and water are mixed evenly, and then mixed with methanol and placed in a water bath to obtain a mixed solution; The mixed solution is uniformly mixed with polyethylene glycol, and sequentially subjected to ultrasonic treatment, crystallization treatment, centrifugation treatment and drying to obtain the β-cyclodextrin metal-organic framework; Mixing 4-hydroxybenzoxazole-2(3H)-thione and acetonitrile to obtain HBOT-acetonitrile solution; The β-cyclodextrin metal organic framework and the HBOT-acetonitrile solution are mixed evenly to obtain the drug delivery system.

7. The preparation method according to claim 6, characterized in that The molar ratio of the β-cyclodextrin to potassium hydroxide is 1:

8.

8. The preparation method according to claim 6, characterized in that The water bath time is 10 min and the temperature is 60°C; The ultrasonic treatment time is 10 minutes and the temperature is 60°C; The crystallization treatment time is 24h; The centrifugal treatment time is 5 min and the rotation speed is 5000 rpm; The drying time is 24 hours and the temperature is 60°C; The mass volume ratio of the β-cyclodextrin metal organic framework and the HBOT-acetonitrile solution is 5 mg:1 mL.

9. Use of the drug delivery system according to claims 2-5 in the preparation of an antibacterial agent, characterized in that: The bacteria targeted by the antibacterial agent include Escherichia coli and / or Shigella flexneri.

10. An antibacterial agent, characterized in that The antibacterial agent comprises the drug delivery system according to claims 2-5; the bacteria targeted by the antibacterial agent include Escherichia coli and / or Shigella flexneri.

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