A method for constructing a novel carrier of a marine source 4-hydroxybenzoxazole-2 (3H) -thione based on beta-cyclodextrin metal organic framework and application of the carrier in enhancing antibacterial activity

Abstract

The present application relates to the field of biological medicine, in particular to a kind of marine source 4-hydroxybenzoxazole-2 (3H) -thione novel carrier construction method based on β-cyclodextrin metal organic framework and its application for enhancing antibacterial activity.The present application finds that 4-hydroxybenzoxazole-2 (3H) -thione (HBOT) can inhibit the growth of escherichia coli and shigella flexneri.The results show that the inhibition diameter of HBOT on shigella flexneri is 15.78±0.67mm, and the inhibition diameter of HBOT on escherichia coli is 10.81±0.50mm;The MIC of free HBOT on shigella flexneri is 128 μg / mL, and the MIC of β-CD-MOF loaded with free HBOT is 64 μg / mL, so β-CD-MOF loaded with free HBOT has the ability to enhance antibacterial performance.

Description

[0001] The present application relates to the field of biological medicine, and particularly relates to a method for constructing a novel carrier of a marine source 4-hydroxybenzoxazole-2(3H)-thione based on a beta-cyclodextrin metal organic framework and application of the carrier in enhancing antibacterial activity. BACKGROUND

[0002] Escherichia coli (E. coli) is a gram-negative short bacillus, peritrichous flagellum, can move, and no spores. It can ferment various sugars to produce acid and gas, and is a normal inhabitant in the intestinal tract of humans and animals. E. coli infects the body through contaminated food or water sources, and after infection, not only can cause diarrhea and food poisoning in the body, but also can lead to serious consequences such as kidney failure.

[0003] Shigella is one of the main pathogenic bacteria of gastrointestinal disease infection. Shigella has four species and at least 47 serotypes, among which Shigella flexneri is the dominant bacteria. Studies have shown that eating food contaminated by Shigella flexneri is the main cause of diarrhea.

[0004] Currently, bacterial contamination is a serious problem in food safety and the food production industry. In the face of the increasing emergence of drug-resistant strains, the use of antibiotics to prevent bacterial contamination is not the best control method. Drug-resistant strains can evolve into new drug-resistant strains in the environment, and the development of antibiotics is far slower than the emergence of drug-resistant strains. Therefore, it is urgent to find a new drug to resist E. coli and Shigella flexneri.

[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 applied to drug loading studies due to the toxicity of the raw materials. In contrast, cyclodextrin metal organic frameworks (beta-CD-MOF) have the advantages of safety, non-toxicity, renewability, environmental friendliness and excellent biocompatibility, and are widely studied as carrier materials. However, the application of beta-CD-MOF in drug delivery systems is limited by its relatively low toxicity, and the synthesis process of beta-CD-MOF is complex and the yield is not ideal. Therefore, it is of great research value to realize the rapid synthesis of beta-CD-MOF by combining it with marine antibacterial agents and to improve its antibacterial efficiency. This method can solve the problems of low yield of beta-CD-MOF and weak antibacterial ability of HBOT, thereby widening its application range in drug sustained release loading. The change of metal center and ligand in MOFs aims to increase its specific surface area (up to 10,000 m 2MOFs have high porosity (up to 50% of the material volume) far exceeding traditional porous materials such as zeolites and activated carbon. The MOF framework is highly functional and can be modified chemically or post-synthetically to achieve diverse structures and applications. The choice of metal and its coordination properties further increases the diversity of MOF structures. The shape and size of MOF channels can be tailored to accommodate different drugs, providing flexibility for drug delivery. Due to their non-toxicity and high drug loading capacity, MOFs are widely used in separation, catalysis, chemical sensing, and drug delivery, showing great potential for scientific and industrial applications. Cyclodextrins (CDs) are natural cyclic oligosaccharides composed of D-glucopyranose units linked by α-1,4-glycosidic bonds. They are classified into α-CD, β-CD, and γ-CD based on the number of glucose units (6, 7, or 8). The height of CDs is approximately 0.8 nm, and the diameters are 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 with a wide upper part and a narrow lower part. With the industrialization of cyclodextrin production, CDs are easily accessible and have "hydrophilic outside and hydrophobic inside" properties, making them highly valuable in food and pharmaceutical industries, especially their unique cavity structure has great potential in drug delivery. β-cyclodextrin metal-organic frameworks (β-CD-MOFs) are porous crystalline materials formed by the coordination of cyclodextrins with alkali metal ions. The traditional synthesis method is gas-phase diffusion. In recent years, new methods such as improved gas-phase diffusion, hydrothermal synthesis, ultrasonic technology, microwave-assisted method, and seed growth technology have been developed. However, these methods face challenges such as complex preparation process and unclear optimal synthesis conditions, limiting their large-scale application. Therefore, exploring the optimal synthesis conditions of β-CD-MOFs is crucial for promoting their widespread application. 4-hydroxybenzoxazole-2(3H)-thione (HBOT) is an isostere of the alkaloid 4-hydroxy-2-benzoxazolone from the mangrove plant Acanthus ilicifolius Linnaeus. However, due to the limited availability and slow growth of Acanthus ilicifolius, the development of its drugs is constrained. Structurally, HBOT is composed of a benzene ring and a five-membered ring containing nitrogen and oxygen heteroatoms, belonging to the category of biologically active compounds. HBOT has low toxicity and certain antibacterial properties, and some of its derivatives have anti-inflammatory effects comparable to traditional anti-inflammatory drugs such as aspirin. The core component of HBOT, benzoxazole-2-thione, also has antibacterial, anti-inflammatory, and analgesic effects. The presence of sulfur atoms enhances its lipophilicity, allowing it to easily penetrate biological membranes and act on cells. To expand its applications, reducing toxicity and further enhancing antibacterial activity are key to expanding its application range. Although research on cyclodextrin-based MOFs and marine-derived antibacterial agents is increasing, there is still a lack of in-depth exploration in integrating both into a unified drug delivery system. Therefore, this invention aims to optimize the synthesis of β-CD-MOFs through the Box-Behnken response surface method and ultrasonic-assisted method to achieve high yield and structural uniformity.This system was used to encapsulate HBOT with limited solubility but antibacterial potential into a layered carrier with enhanced porosity and pH-responsive release behavior. To the best of our knowledge, this is the first study on the hybridization of beta-cyclodextrin with MOFs for the controlled delivery of HBOT through green synthesis and comprehensive kinetic modeling, cell compatibility analysis, and antibacterial verification of gram-negative strains. This method provides a novel and scalable approach for developing biocompatible and pH-responsive antibacterial carriers. SUMMARY

[0006] The purpose of the present application is to provide a novel carrier construction method based on beta-cyclodextrin metal organic framework of marine source 4-hydroxybenzoxazole-2(3H)-thione and its application for enhancing antibacterial activity, in order to solve the problems existing in the prior art.

[0007] To achieve the above purpose, the present application provides the following solutions:

[0008] The present application provides the application of 4-hydroxybenzoxazole-2(3H)-thione in the preparation of a drug for resisting Escherichia coli or Shigella flexneri.

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

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

[0011] Beta-cyclodextrin, potassium hydroxide and water are uniformly mixed, then mixed with methanol, and subjected to water bath to obtain a mixed solution;

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

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

[0014] Preferably, the water bath is performed for 10 min at 60 DEG C;

[0015] The ultrasonic treatment is performed for 10 min at 60 DEG C;

[0016] The crystallization treatment is performed for 24 h;

[0017] The centrifugal treatment is performed for 5 min at a speed of 5000 rpm;

[0018] The drying is performed for 24 h at 60 DEG C.

[0019] The application provides a preparation method of the drug delivery system, and the preparation method comprises the following steps:

[0020] The beta-cyclodextrin, potassium hydroxide and water are uniformly mixed, and then mixed with methanol, and then water bath is performed to obtain a mixed solution;

[0021] The mixed solution is uniformly mixed with polyethylene glycol, and then sequentially subjected to ultrasonic treatment, crystallization treatment, centrifugal treatment and drying to obtain the beta-cyclodextrin metal organic framework.

[0022] The 4-hydroxybenzoxazole-2(3H)-thione and acetonitrile are mixed to obtain an HBOT-acetonitrile solution;

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

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

[0025] Preferably, the water bath is performed for 10 min at 60 DEG C.

[0026] The ultrasonic treatment is performed for 10 min at 60 DEG C.

[0027] The crystallization treatment is performed for 24 h.

[0028] The centrifugal treatment is performed for 5 min at a speed of 5000 rpm.

[0029] The drying is performed for 24 h at 60 DEG C.

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

[0031] The application provides an application of the drug delivery system in preparation of an antibacterial agent, and the antibacterial agent is directed against Escherichia coli or Shigella flexneri.

[0032] The application provides an antibacterial agent, which comprises the drug delivery system, and the antibacterial agent is directed against Escherichia coli or Shigella flexneri.

[0033] The application discloses the following technical effects:

[0034] The present application finds that 4-hydroxybenzoxazole-2(3H)-thione has an antibacterial effect on E. coli or Shigella flexneri. The present embodiment evaluates the inhibition zone diameter and MIC of 4-hydroxybenzoxazole-2(3H)-thione by the Oxford cup method. The results show that the inhibition zone diameter of 4-hydroxybenzoxazole-2(3H)-thione on Shigella flexneri is 15.78±0.67 mm, and the inhibition zone diameter on E. coli is 10.81±0.50 mm; the MIC of free 4-hydroxybenzoxazole-2(3H)-thione on Shigella flexneri is 128 μg / mL, and the MIC of β-CD-MOF loaded with free 4-hydroxybenzoxazole-2(3H)-thione is 64 μg / mL. It can be seen that β-CD-MOF loaded with free 4-hydroxybenzoxazole-2(3H)-thione has the ability to enhance the antibacterial performance.

[0035] 4-hydroxybenzoxazole-2(3H)-thione (HBOT) is an isostere of the alkaloid 4-hydroxy-2-benzoxazolone in mangrove plants Acanthus ilicifolius Linnaeus. However, due to its limited source and slow growth, the research and development of its biological activity is restricted. In order to reduce the toxicity of 4-hydroxybenzoxazole-2(3H)-thione and further improve the antibacterial effect of 4-hydroxybenzoxazole-2(3H)-thione, the present application provides a drug delivery system (HBOT / β-CD-MOF or HBOT / β-CD-MOF complex), which comprises a carrier loaded with 4-hydroxybenzoxazole-2(3H)-thione, and the carrier is a β-cyclodextrin metal organic framework. The present application integrates β-cyclodextrin (β-CD) into the structure of metal organic framework (MOF) (i.e. β-CD-MOF), and a biocompatible high-efficiency carrier is prepared, which has enhanced physicochemical properties and is suitable for pH-responsive drug delivery. The HBOT / β-CD-MOF complex shows excellent antibacterial performance on Shigella flexneri and E. coli, and the inhibition zone reaches 21.54±0.4 mm, which is significantly better than free HBOT through drug release effect. The CCK-8 cytotoxicity experiment confirms its high biocompatibility, and the cell survival rate is more than 103% at a concentration of 800 μg / mL.

[0036] In specific embodiments, the beta-CD-MOF is synthesized by an ultrasonic-assisted method and optimized by Box-Behnken response surface method to maximize the yield and structural performance. The results show that the solvent volume and reaction temperature are the key variables, and when the molar ratio is 8:1 (KOH: beta-CD), the solvent is 20 mL, and the temperature is 60°C, the optimal yield reaches 82.39%. Subsequently, the scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and nitrogen adsorption-desorption isotherm characterization confirm that the beta-CD-MOF is successfully formed with a high surface area (332.13 m 2 / g) of a layered structure, and after encapsulating 4-hydroxybenzoxazole-23H)-thione (HBOT), the surface area slightly decreases to 247.07 m 2 / g; in vitro release studies show that it has pH-dependent release kinetics: under pH 2 conditions, it is mainly dominated by first-order diffusion (R 2 = 0.977), while under pH 7 conditions, due to the combined action of swelling and diffusion mechanisms, it conforms to the Ritger-Peppas equation description of non-Fickian diffusion (R 2 = 0.97, n = 0.70); the HBOT / beta-CD-MOF complex exhibits excellent antibacterial performance against Shigella flexneri or Escherichia coli; it is significantly better than free HBOT through the drug release effect; the CCK-8 cell toxicity experiment confirms its high biocompatibility, and the cell survival rate is more than 103% at a concentration of 800 μg / mL. Overall, the present application provides an expandable environmentally friendly method for subsequent research, which is used to develop a multifunctional MOF-based carrier with responsive release characteristics, and provides promising potential for safer, long-acting, and targeted antibacterial therapy. BRIEF DESCRIPTION OF DRAWINGS

[0037] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed in the embodiments will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without creative labor.

[0038] Figure 1 Preparation process of HBOT / beta-CD-MOF;

[0039] Figure 2 Response surface and contour plots of the effects of the interaction of the molar ratio of raw materials and the amount of methanol (A), the molar ratio of raw materials and ultrasonic temperature (B), and the amount of methanol and ultrasonic temperature (C) on the yield of beta-CD-MOF;

[0040] Figure 3Scanning electron microscope 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; wherein, A is the comparison of swelling behavior of HBOT / β-CD-MOF and β-CD-MOF in neutral buffer (25℃, pH 7.0); B is the adsorption kinetics curve under different HBOT concentrations; C is the equilibrium isotherm under different HBOT concentrations; D-I are the adsorption mechanism analysis of pseudo-first order, pseudo-second order, Elovich, Scatchard, Langmuir and Freundlich models, respectively; J is the N2adsorption-desorption isotherm for porosity analysis; K and L are the in vitro release profiles of artificial gastric fluid (AGF, pH 2.0) and artificial intestinal fluid (AIF, pH 7.0), respectively, showing pH-dependent slow release;

[0043] Figure 6 Detection of HBOT-β-MOF in AGF (1h, a; 3h, c; 6h, e) and AIF (1h, b; 9h, d; 20h, f) at different times by high performance liquid chromatography;

[0044] Figure 7 Effect of HBOT, β-CD-MOF and HBOT / β-CD-MOF on the viability of MODE-K cells; wherein, A is the no sample control; B is 800 μg / mL β-CD-MOF; C is 800 μg / mL HBOT / β-CD-MOF; D is the effect of different substances at different concentrations on cell survival rate;

[0045] Figure 8A is the antibacterial activity evaluation of HBOT / β-CD-MOF on Shigella flexneri, 1 is a blank control (0 μg / mL), 2 is β-CD-MOF, 3 is HBOT / β-CD-MOF, 4 is free HBOT, 5 is a positive control (vancomycin 0.01 μg / mL); B is the antibacterial activity evaluation of HBOT / β-CD-MOF on Escherichia coli, 1 is a blank control (0 μg / mL), 2 is β-CD-MOF, 3 is HBOT / β-CD-MOF, 4 is free HBOT, 5 is a 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 antibacterial rate statistical result of HBOT / β-CD-MOF on Salmonella flexneri (n = 3; #p < 0.05, ##p < 0.01 vs free HBOT). DETAILED DESCRIPTION

[0046] Various exemplary embodiments of the present application will now be described in detail with reference to the drawings. The detailed description is not to be considered to limit the application in any way, but rather to illustrate certain aspects, features and embodiments of the application.

[0047] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of the term "about" in relation to a value or a range of values is intended to include each individual intermediate value and each smaller range that falls within the range of values. The upper and lower limits of these smaller ranges can be included or excluded from the range.

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, preferred methods and materials are described. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any reference is not an admission that it is prior art with respect to the present application. All documents mentioned herein are incorporated herein by reference.

[0049] Many modifications and variations of this application can be made in the light of the above teachings without departing from the spirit and scope thereof. Other implementations of this application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. The examples and embodiments described herein are exemplary only and are not intended to be limiting.

[0050] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” and the like are open-ended terms that mean inclusion, but not limited to.

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

[0052] (I) Experimental Methods

[0053] 1. Chemical Reagents

[0054] 4-Hydroxybenzoxazole-2(3H)-thione (HBOT, purity > 98%, verified by high performance liquid chromatography) was purchased from China Institute for Food and Drug Control (Beijing). β-Cyclodextrin (β-CD, purity > 98.0%) and potassium hydroxide (KOH, purity > 99.0%) were purchased from Shanghai Maikelin 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 pure, purity > 98%) was purchased from Beijing Chemical Reagent Factory.

[0055] 2. Instruments and Equipment

[0056] The framework material was characterized by Fourier transform infrared spectroscopy (FT-IR; PU9800, Panalytical, Almelo, Netherlands). The surface morphology was observed by scanning electron microscopy (SEM, Gemini SEM-300 microscope, Zeiss, Oberkochen, Germany) with an instrument working voltage of 3 kV, which could capture clear and complete surface images. The quantitative analysis of HBOT was performed using a Waters E2695+UV high performance liquid chromatograph. The product was synthesized by a CNC temperature-variable ultrasonic cleaner (KQ250DE, 4 kHz) from Shanghai Jingqi Instrument Co., Ltd., which was used for ultrasonic and thermal treatment. 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 292 nm). Separation was performed using a C 18 chromatographic column (COSMOSIL C 18 18 ODS-3, 2.1 mm x 150 mm, 5 μm) at a column temperature of 25°C. The mobile phase was acetonitrile (A) and ultrapure water (B), with a sample injection volume of 20 μL and a flow rate of 1 mL / min. Data analysis and graphics processing were performed using Design-Expert 13.0 software (Stat-Ease, USA). The E. coli model was ATCC 25922, and the Shigella flexneri model was ATCC 12022, both of which were donated by 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 framework

[0058] The preparation process is shown in Figure 1 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 then the filtrate was added to form a milky white solution. The mixture was placed in a 60°C water bath for heating for 10 min until a clear solution was obtained. Subsequently, the solution was transferred to an ultrasonic cleaner (4000 Hz) at different temperatures. Before ultrasonic treatment, polyethylene glycol-8000 (PEG-8000, 108 mg) was added, and the solution was ultrasonically treated for 10 min. After that, the solution was placed at room temperature and transferred to a 50 mL centrifuge tube for crystallization for 24 h. The crystallized solution was centrifuged at 5000 rpm for 5 min, and the resulting product was dried in a 60°C constant temperature oven for 24 h. After cooling to room temperature, the β-cyclodextrin metal organic framework (β-CD-MOF) material was obtained by ultrasound-assisted preparation. Subsequently, HBOT was loaded onto the β-CD-MOF to prepare the HBOT / β-CD-MOF composite by different concentration gradients (see Step “Dynamic and static adsorption test” for details).

[0059] 3. Single factor experiment

[0060] The controlled variable method was used to keep other factors unchanged and evaluate the influence of a single variable on the experimental results. The factors affecting the yield of β-CD-MOF, including the molar ratio of KOH to β-CD, the amount of methanol, and the ultrasonic temperature, were selected for single factor experiment to evaluate the influence of different levels of each factor on the yield of β-CD-MOF. Subsequently, the three factors with the most significant influence were selected for Box-Behnken response surface experiment. Since the theoretical mass of CD-MOF material cannot be calculated, the mass of β-CD was used as a substitute for the theoretical mass, and the following yield formula was obtained:

[0061] Yield (%) = m1 / m2 x 100%;

[0062] wherein 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). Using the Box-Behnken response surface methodology, a three-factor, three-level experimental model was established with the β-CD-MOF yield as the response variable. The molar ratios (A) were 6:1, 8:1, and 10:1 (corresponding to -1, 0, and +1 levels), the solvent volumes (B) were 15, 20, and 25 mL (corresponding to -1, 0, and +1 levels), and the reaction temperatures (C) were 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] Dry samples of HBOT / β-CD-MOF and β-CD-MOF were immersed in a 0.9% NaCl solution at 37°C. Samples were removed every 10 minutes, surface moisture was gently absorbed, and the samples were weighed. This process lasted for 120 minutes. The swelling ratio was calculated at each time point to assess the swelling behavior. Swelling kinetics analysis revealed the rapid swelling of β-CD-MOF, attributed to its high permeability, and the stable saturation swelling curves of HBOT / β-CD-MOF. The swelling ratio was calculated using the following formula:

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

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

[0069] 7. Dynamic and static adsorption tests

[0070] Two centrifuge tubes were each filled with 5 mg of β-CD-MOF. One tube was injected with 1 mL of HBOT-acetonitrile solution of different concentrations (10, 30, 50, 100, 250, 500, 800, and 1000 μg / mL) (HBOT-acetonitrile solution was prepared by mixing different masses of 4-hydroxybenzoxazole-2(3H)-thione and acetonitrile), and 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 capacity (Q (μg / mg)) was determined by the following formula:

[0071] Q = (C0 - Ce) / m × V;

[0072] Wherein, Co 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 the 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] In the formula, 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] In the formula, 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] In the formula, Q max is the maximum saturated adsorption amount, and Ks is the dissociation constant.

[0085] Langmuir model:

[0086]

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

[0088] Freundlich model:

[0089]

[0090] In the formula, 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] The 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 (about 77 K) and the adsorption amount at different pressures was measured.

[0093] 9. In vitro sustained release experiment

[0094] The 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 the previously prepared HBOT / β-CD-MOF was added to 10 mL of AGF or AIF and fully shaken in a constant temperature water bath shaker at 37°C and 180 rpm. The reaction in AGF lasted for 6 h, and 1 mL of supernatant was collected by centrifugation every hour, then 1 mL of fresh AGF was added to the system. In contrast, the reaction in AIF was extended to 36 h, and 1 mL was sampled at 1, 3, 6, 9, 18, and 24 h, respectively, and 1 mL of fresh AIF was added to the reaction system after each sampling. The cumulative release rate (DS%) of HBOT was calculated according to the following formula:

[0095]

[0096] In the formula, 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 released medium at the i th time, V o represents the total volume of the released medium, C n represents the concentration of the released medium, M ∞ represents the content of HBOT in HBOT / β-CD-MOF. The experiment was carried out for three times, and the results were analyzed by taking the average value.

[0097] 10. In vitro drug release kinetics

[0098] The drug release process of HBOT / β-CD-MOF was analyzed by using various kinetic models. The relevant model equations are as follows:

[0099] D t = Kt + D0;

[0100] D t is the cumulative release rate of drug 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] D t is the cumulative release rate of 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] D t is the release amount of drug at time t, and K h is the release rate constant.

[0105] D t = K p t n ;

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

[0107]

[0108] D t represents the proportion of the release 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 seeded in a 96-well plate at a density of 1 × 10 3 cells per well, and were 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, and the loading rate was 27.99%), and a β-CD-MOF control group receiving an equivalent carrier dose was set. After 24 h of incubation, 10 μL of CCK-8 solution was added to each well, and incubation was continued at 37°C in 5% CO2 for 2 h. Subsequently, the absorbance was measured at 490 nm by a microplate reader, and the cell survival rate was calculated to evaluate the cytotoxicity. Note that only the final concentration of HBOT is indicated in the experimental data, and the same indication method is used in the antibacterial experiment. The cell survival rate calculation formula is as follows:

[0111]

[0112] wherein, G is the average value of the normal control group, and n G is the average value of the normal control group, and

[0113] 12. Minimum inhibitory concentration (MIC) of the compound against bacteria

[0114] Oxford cup method was used to evaluate the diameter of the inhibition zone. The specific steps were as follows: 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 into independent oxford cups, respectively. The plates were incubated at 37°C under controllable temperature and humidity for 24 h. 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 E. coli and S. flexneri was quantitatively detected by micro-broth double dilution method. The bacterial suspension with a turbidity equivalent to 0.5 MacFarland standard was prepared and inoculated into 96-well plates. 0.1% vancomycin was used as a positive control (PC), and untreated blank wells were used as a negative control (BC). HBOT was serially diluted from 128 μg / mL to 1 μg / mL, while 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 bacterial growth was monitored by spectrophotometry at 600 nm (OD 600 ) and the minimum inhibitory concentration (MIC) was determined. The antibacterial effect was calculated by the following formula:

[0116]

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

[0118] (B) Results

[0119] 1. Single factor synthesis condition optimization of β-CD-MOF

[0120] Porous MOF materials have realized sustained and efficient delivery of therapeutic agents for drug controlled release, thereby expanding the functional range of MOFs in biomedical applications. Here, the Box-Behnken response surface method was used to optimize the synthesis parameters. The statistical analysis of the model showed that the volume of the solvent (factor B) and its quadratic term B 2 and C 2The β-CD-MOF yield was significantly affected by the molar ratio of KOH to β-CD (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%, as shown in Figure 2 .

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

[0122] The three main factors affecting β-CD-MOF were the molar ratio of raw materials, methanol volume fraction, and ultrasonic temperature. The multivariate quadratic regression equation for yield was fitted by experimental data as follows:

[0123] Y = -466.914 + 7.3273A + 15.587B + 11.5003C + 0.647.AB - 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 under 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%); meanwhile, 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 changed from a dense form to a relatively loose layered structure after loading HBOT (A and C in Figure 3 The inherent flaky structure of β-CD-MOF (B and D in Figure 3 ) and the lack of strong covalent bonds between β-CD and K + may have caused a columnar displacement of β-CD-MOF after HBOT encapsulation, leading to loose arrangement. Due to the high polarity of HBOT, its direct adsorption capacity by β-CD was limited, suggesting that the observed layered structure was not only related to physical aggregation, but also involved a structural transformation of the MOF itself. The ionic interaction between β-CD and K + may have promoted the formation of this layered framework. This unique structure facilitated 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, effectively trapping 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 As shown in Figure A, the characteristic absorption band of KOH is 3583 cm⁻¹. -1 (-OH stretching vibration). The characteristic peak of β-CD is located at 3591.9 cm⁻¹. -1 1623.2cm -1 and 1137.2cm -1 There are four distinct characteristic absorption bands in β-CD-MOF, among which 2931m -1 The CH stretching vibrations at position -CH3 and -CH2 are observed at 1612.2 cm⁻¹. -1 and 1147.3cm -1 The peak indicates CC, CCO, or CCO stretching vibrations. The infrared spectra of β-CD-MOF and β-CD show no significant difference, indicating the presence of a β-CD framework in the synthesized material. 3611 cm- -1 The blue shift of the 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. HBOT at 1616 cm⁻¹ -1 and 1504cm -1 The peak at this point is attributed to the stretching vibration of the benzene ring skeleton; the CH stretching vibration peak is located at 1265 cm⁻¹. -1 The -C=S stretching vibration peak is located at 1002 cm⁻¹. -1 The hydroxyl absorption peak of HBOT / β-CD-MOF red-shifted to 3452 cm⁻¹. -1 The increased frequency suggests an interaction between the OH group of β-CD-MOF and HBOT, indicating the successful synthesis of HBOT / β-CD-MOF.

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

[0130] To further investigate intermolecular interactions, the XRD patterns of KOH, β-CD-MOF, and HBOT / β-CD-MOF are shown below. Figure 4 As shown in Figure B, the 12-30° peaks are characteristic diffraction peaks of KOH and β-CD-MOF. The spectrum of β-CD-MOF shows several diffraction peaks slightly shifted, disappearing, or replaced by clearer characteristic peaks, indicating that β-CD reacted with KOH during the synthesis process. + The interactions resulted in a more ordered crystal structure. Furthermore, loading HBOT onto β-CD-MOF did not alter the crystalline state of β-cyclodextrin.

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

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

[0133] 7. Swelling performance

[0134] As shown in A of Figure 5 , the swelling rates of HBOT / β-CD-MOF and β-CD-MOF changed with time, and both showed a trend of first increasing and then decreasing after 100 min. Notably, the swelling behavior of HBOT / β-CD-MOF gradually stabilized after reaching a more obvious saturation state. In contrast, β-CD-MOF crosslinked with HBOT molecules showed a higher swelling rate due to higher permeability and solvent absorption capacity, indicating that β-CD-MOF had a stronger 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 B and C of Figure 5 , respectively. In the dynamic process, the adsorption effect was significant within the initial 300 min; in the static adsorption, β-CD-MOF reached a saturation state at a concentration of 800 μg / mL, with a drug loading rate of 27.99 ± 3.12%. The adsorption kinetics and isotherm, dynamic fitting pseudo-first-order and pseudo-second-order equations, and Elovich plots are shown in D-F of Figure 5 ; the static fitting Scatchard, Langmuir, and Freundlich equations are shown in G-I of Figure 5 , and the corresponding parameters are shown in Table 1. The pseudo-first-order model is suitable for the adsorption behavior at a higher adsorbent concentration, 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 are more consistent with the pseudo-first-order model (R 2>0.99), which is suitable for saturated homogeneous adsorbents (such as molecularly imprinted polymers), while the pseudo-second order model assumes that adsorption occurs on a heterogeneous surface with interactions between adsorbed molecules. The Scatchard model plays an important role in the theory of regular solution and non-electrolyte solution, and the Langmuir equation assumes that adsorption occurs on a heterogeneous surface through intermolecular interactions, which is more suitable for static adsorption conditions. The results show 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] 9. Nitrogen adsorption-desorption isotherm analysis

[0140] 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 J in Figure 5 Both materials form stacking pores in a sheet-like structure. Compared with β-CD-MOF, the specific surface area and pore volume of HBOT / β-CD-MOF gradually increase, the hysteresis loop improves the mesopore regularity, but the pore size gradually decreases and the crystallinity improves after loading HBOT. The Brunauer-Emmett-Teller (BET) values of HBOT / β-CD-MOF and β-CD-MOF are 247.0650 m 2 / g and 332.1285 m 2 / g, respectively, indicating that β-CD-MOF is sufficient to adsorb HBOT.

[0141] 10. In vitro sustained-release ability

[0142] As shown in K in Figure 5 , 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 is 250 min, and then gradually decreases, with a slower rate, indicating that the drug is decomposed. For AIF, the cumulative drug release increases rapidly within 10 h, with a faster release rate in the first 1-10 h, and then gradually slows down, showing good sustained-release effect (L in Figure 5 ). The release concentration of HBOT / β-CD-MOF in AIF and AGF was determined by HPLC at different time intervals (M in 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 acidic gastric juice with low pH may destroy the structure of HBOT / β-CD-MOF, while the weak alkaline intestinal fluid is more conducive to the slow release of the drug.

[0143] 11. Control release mechanism

[0144] Various mathematical models, including zero-order, first-order, Higuchi, Ritger-Peppas, Redlich-Peterson, and Weibull, were employed to evaluate the release kinetics and potential mechanisms of HBOT from HBOT / β-CD-MOF under different pH conditions (Table 2). The results showed that pH significantly affected the drug release behavior: under acidic conditions (pH = 2), the release profile best fit the first-order kinetic model (R2= 0.977), indicating that drug diffusion was mainly driven by the concentration gradient; under neutral conditions (pH = 7), the Ritger-Peppas model (R2= 0.974; release exponent n = 0.7018) revealed a non-Fickian diffusion mechanism, possibly due to the combined action of carrier swelling and diffusion processes. The Higuchi model had a poor fit under both pH conditions (R2< 0.83), suggesting that simple diffusion was not the main release mechanism. In addition, the Weibull model had a strong correlation at pH = 7 (R2= 0.952), supporting the existence of a more complex pH-responsive release pathway, possibly related to structural changes in the MOF matrix. These findings revealed the multi-mechanism nature of drug release, providing a theoretical basis for designing pH-sensitive delivery systems with adjustable release profiles. 2 2 2 2

[0145] Table 2. Drug release kinetic model parameters.

[0146]

[0147] 12. Cell activity impact

[0148] The results of CCK-8 method for detecting cell growth and survival rate are shown in Figure 7 . The blank control group of mouse intestinal epithelial cells showed a single layer of non-overlapping spindle-shaped cells (A in Figure 7 ); the 800 μg / mL HBOT treatment group showed cell fragmentation and contraction (B in Figure 7 ), while the above phenomena were significantly alleviated after HBOT / β-CD-MOF pretreatment (C in Figure 7 ); the β-CD-MOF group had no effect on cell growth (D in Figure 7 ). The 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 low (only 72.76 ± 3.46%), and the cell growth of the HBOT / β-CD-MOF group at this concentration was significantly better than that of the HBOT group (P < 0.01), again confirming its safety as a drug carrier.​​​​Figure 7 (E in the text). The results also showed that HBOT / β-CD-MOF had a protective effect on cells, ultimately resulting in an average cell viability greater than 100%. Interestingly, HBOT could still stimulate cell growth at concentrations below 400 μg / mL, but cytotoxicity was significant above 400 μg / mL. The sustained-release properties of HBOT / β-CD-MOF effectively improved the antibacterial safety of HBOT by maintaining cells in an environment with suitable drug concentrations.

[0149] 13. Evaluation of antibacterial effect

[0150] like Figure 8 As shown in A and B, β-CD-MOF had no antibacterial effect against either Shigella flexneri or Escherichia coli. HBOT showed an inhibition diameter of 15.78±0.67 mm against Shigella flexneri and 10.81±0.50 mm against Escherichia coli. The antibacterial effect of HBOT / β-CD-MOF was significantly better than that of free HBOT (P<0.01). Its inhibition diameter against Shigella flexneri was 21.54±0.43 mm, and against Escherichia coli it was 18.45±0.46 mm, with a stronger inhibitory effect against the former (P<0.01). Figure 8 (C in the text). Further research shows ( Figure 8 In the study (D), the inhibitory activity (MIC) of HBOT against Shigella flexneri was 128 μg / mL, while that of HBOT / β-CD-MOF was 64 μg / mL. The inhibitory activity of HBOT / β-CD-MOF against Shigella flexneri was superior to that of HBOT (P≤0.05). This may be attributed to the sustained-release effect, which prolongs the interaction time between HBOT and bacteria, indicating that HBOT / β-CD-MOF can be used as a novel antibacterial agent.

[0151] (III) Conclusion

[0152] The application successfully synthesizes beta-cyclodextrin metal organic framework (beta-CD-MOF) by ultrasonic-assisted method, and optimizes the conditions by Box-Behnken response surface method, and the yield reaches 82.39% under ideal parameters. The composite material effectively encapsulates marine-derived compound 4-hydroxybenzoxazol-2(3H)-thione (HBOT), forms a layered structure with good porosity and specific surface area, and realizes high drug loading efficiency. Compared with traditional cyclodextrin or MOF-based carriers, the hybrid system shows enhanced pH-responsive release behavior, and can realize sustained and controllable delivery of drugs under intestinal conditions. Importantly, HBOT / beta-CD-MOF exhibits better antibacterial activity than free HBOT against Shigella flexneri and Escherichia coli, and excellent biocompatibility. The combination of safe materials, scalable green synthesis process and environmental stability highlights the application potential of the system in the field of biomedical and pharmaceutical in the future. Compared with the previous work of studying MOF or cyclodextrin system alone, the application constructs a synergistic carrier with drug stability, release kinetics and biological activity by structural integration, and detailed kinetic modeling and adsorption analysis provide new insights into the interaction mechanism of drug-carrier in variable pH environment.

[0153] The above-described embodiments are only used to describe the preferred modes of the present application, and do not limit the scope of the present application, and various modifications and improvements of the technical solutions of the present application made by those skilled in the art without departing from the design spirit of the present application shall fall within the protection scope of the present application.

Claims

1. Use of 4-hydroxybenzoxazole-2(3H)-thione in the preparation of a medicament for the treatment of E. coli or Shigella flexneri.

2. A drug delivery system against E. coli or Shigella flexneri, characterized in that, The drug delivery system comprises a carrier loaded with 4-hydroxybenzoxazole-2(3H)-thione; the carrier is a β-cyclodextrin metal organic framework; The preparation method of the β-cyclodextrin metal organic framework comprises the following steps: The β-cyclodextrin, potassium hydroxide and water are uniformly mixed, then mixed with methanol, and subjected to water bath to obtain a mixed solution; The mixed solution is uniformly mixed with polyethylene glycol, and sequentially subjected to ultrasonic treatment, crystallization treatment, centrifugal treatment and drying to obtain the β-cyclodextrin metal organic framework; The molar ratio of the β-cyclodextrin to potassium hydroxide is 1:8; The water bath is performed for 10 min at 60°C; The ultrasonic treatment is performed for 10 min at 60°C; The crystallization treatment is performed for 24 h; The centrifugal treatment is performed for 5 min at a speed of 5000 rpm; The drying is performed for 24 h at 60°C.

3. The method of making a drug delivery system of claim 2, characterized in that, The preparation method comprises the following steps: The β-cyclodextrin, potassium hydroxide and water are uniformly mixed, then mixed with methanol, and subjected to water bath to obtain a mixed solution; The mixed solution is uniformly mixed with polyethylene glycol, and sequentially subjected to ultrasonic treatment, crystallization treatment, centrifugal treatment and drying to obtain the β-cyclodextrin metal organic framework; The 4-hydroxybenzoxazole-2(3H)-thione and acetonitrile are mixed to obtain an HBOT-acetonitrile solution; The β-cyclodextrin metal organic framework and the HBOT-acetonitrile solution are uniformly mixed to obtain the drug delivery system The molar ratio of the β-cyclodextrin to potassium hydroxide is 1:8; The water bath is performed for 10 min at 60°C; The ultrasonic treatment is performed for 10 min at 60°C; The crystallization treatment is performed for 24 h; The centrifugal treatment is performed for 5 min at a speed of 5000 rpm; The drying is performed for 24 h at 60°C.

4. Use of the drug delivery system according to claim 2 for the manufacture of an antibacterial agent, characterized in that The mass-volume ratio of the β-cyclodextrin metal organic framework to the HBOT-acetonitrile solution is 5 mg:1 mL.

5. An antibacterial agent, characterized by, The antibacterial agent is directed against E. coli or Shigella flexneri. The antibacterial agent comprises the drug delivery system of claim 2; and the antibacterial agent is directed against E. coli or Shigella flexneri.

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