Pyridone carboxyl derivatives and acid-degraded organometallic framework compositions and their application in Helicobacter pylori

Abstract

This invention provides a composition of a pyridone carboxyl derivative and an acid-degraded organometallic framework, and its application in Helicobacter pylori. Specifically, it provides the application of the pyridone carboxyl derivative and the acid-degraded organometallic framework composition in the preparation of delivery carriers or drugs for the treatment / prevention of Helicobacter pylori infection. The composition of the pyridone carboxyl derivative and the acid-degraded organometallic framework can serve as a carrier for carrying singlet oxygen. Under acidic conditions, it degrades and releases metal ions with bactericidal functions. It can be used for bacterial infections in acidic, anaerobic, and light-free environments, such as Helicobacter pylori infection in the stomach. The released metal ions and singlet oxygen enter the cell, promoting the leakage of cell contents and interfering with the metabolism of bacteria such as Helicobacter pylori. The combined effect of both exhibits good antibacterial ability.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a composition and its use in the preparation of a delivery carrier or medicament for the treatment / prevention of Helicobacter pylori infection. Background Technology

[0002] Helicobacter pylori is a Gram-negative spiral-shaped bacterium that colonizes the stomach. By releasing urease to break down urea and produce NH3 to neutralize stomach acid, Helicobacter pylori can combat the acidic environment of the stomach. It then migrates to the gastric mucosa via flagella, secreting adhesins to adhere to epithelial cells. This mechanism allows Helicobacter pylori to persistently colonize the human gastric mucosa and cause chronic gastric infection. Helicobacter pylori is primarily transmitted through the oral-oral or fecal-oral route and can lead to diseases such as chronic gastritis, peptic ulcers, gastric cancer, and gastric mucosa-associated lymphoid tissue lymphoma. Furthermore, recent studies have found a close association between Helicobacter pylori infection and certain non-gastrointestinal diseases, such as unexplained iron deficiency anemia and immune thrombocytopenic purpura. In 2017, the World Health Organization classified Helicobacter pylori (infection) as a Group 1 carcinogen.

[0003] Therefore, the current clinical practice recommendation for Helicobacter pylori infection is to eradicate it once it is detected. However, current treatments for Helicobacter pylori have problems such as drug resistance, multiple drug combinations, high dosage, and significant side effects. Summary of the Invention

[0004] In view of this, in order to at least partially solve at least one of the aforementioned technical problems, the present invention provides a pyridone carboxyl derivative and an acid-degraded organometallic framework composition and its application in Helicobacter pylori.

[0005] According to one embodiment of the present invention, a composition is provided comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework.

[0006] According to one embodiment of the present invention, the metal node material of the acid-degraded organometallic framework includes at least one of silver, zinc, and nickel.

[0007] According to one embodiment of the present invention, the acid-degraded organometallic framework is a zinc-based zeolite-imidazolium ester framework material.

[0008] According to one embodiment of the present invention, the pyridone derivative comprises the structure shown in formula (I):

[0009] (I).

[0010] According to one embodiment of the present invention, the composition further comprises a singlet oxygen molecule carried therein, preferably, the composition is a nanomedicine.

[0011] According to another embodiment of the present invention, a method for preparing a composition is provided, comprising:

[0012] Adding a photosensitizer to a pyridone carboxyl derivative and then introducing oxygen yields a pyridone derivative carrying a singlet oxygen molecule.

[0013] The pyridone carboxyl derivative carrying singlet oxygen molecules is combined with an organometallic framework through adsorption to obtain a composition with a core-shell structure.

[0014] According to one embodiment of the present invention, the photosensitizer includes at least one of dihydroporphyrin E6, methylene blue, or indocyanine green.

[0015] According to another aspect of the present invention, the use of a composition in the preparation of a delivery carrier or medicament for the treatment / prevention of Helicobacter pylori infection is provided.

[0016] According to another aspect of the present invention, a pharmaceutical composition is provided, comprising the composition and a pharmaceutically acceptable carrier thereof.

[0017] According to one embodiment of the present invention, the pharmaceutical composition is in a dosage form suitable for oral administration.

[0018] According to embodiments of the present invention, a composition comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework can serve as a carrier for singlet oxygen. Under acidic conditions, it degrades and releases metal ions with bactericidal functions, while continuously releasing singlet oxygen independent of oxygen and light. It can be used for bacterial infections in acidic, hypoxic, and light-free environments, such as Helicobacter pylori infection in the stomach. The released metal ions and singlet oxygen enter the cell, promoting the leakage of cell contents and interfering with the metabolism of bacteria such as Helicobacter pylori. The combined effect of both exhibits good antibacterial ability. Attached Figure Description

[0019] The above and other objects, features and advantages of the present invention will become more apparent from the following description of embodiments of the invention with reference to the accompanying drawings, in which:

[0020] Figure 1 This invention relates to Py-COOH- 1 Schematic diagram of O2@ZIF-8 preparation;

[0021] Figure 2 The following figures represent the characterization results of Py-COOH@ZIF-8 in the embodiments of the present invention: A is a transmission electron microscope image of Py-COOH@ZIF-8 under neutral conditions, B is a transmission electron microscope image of Py-COOH@ZIF-8 under acidic conditions, and C is a zeta potential diagram.

[0022] Figure 3 The Py-COOH- of this embodiment of the invention 1 UV curves of loading and releasing singlet oxygen in O2@ZIF-8;

[0023] Figure 4 The Py-COOH- of this embodiment of the invention 1 Electron spin resonance spectrum of loaded and released singlet oxygen in O2@ZIF-8;

[0024] Figure 5 This invention relates to Py-COOH- 1 Figure showing the in vitro antibacterial and bacteriostatic rate results of O2@ZIF-8;

[0025] Figure 6 This invention relates to Py-COOH- 1 Image of O2@ZIF-8 in vitro antibacterial plating plate and scanning electron microscopy characterization results;

[0026] Figure 7 This is an image showing the HE staining results of an in vivo antibacterial experiment in mice according to an embodiment of the present invention;

[0027] Figure 8 This invention relates to Py-COOH- 1 Figure showing the results of the O2@ZIF-8 hemolysis experiment;

[0028] Figure 9 This invention relates to Py-COOH- 1 Figure showing the results of the O2@ZIF-8 cell compatibility experiment;

[0029] Figure 10 This invention relates to Py-COOH- 1 O2@ZIF-8 biosafety histological staining results;

[0030] Figure 11 This invention relates to Py-COOH- 1 The results of the O2@ZIF-8 biosafety mouse gut microbiota analysis are shown in Figure A, which is a bar chart of the abundance of mouse fecal microbiota; Figure B is a heatmap of the abundance of mouse fecal microbiota; and Figure C is a graph showing the results of the functional prediction analysis of mouse gut microbiota. Detailed Implementation

[0031] The embodiments of the present invention will now be described with reference to the accompanying drawings. However, it should be understood that these descriptions are exemplary only and are not intended to limit the scope of the invention. In the following detailed description, numerous specific details are set forth to provide a comprehensive understanding of the embodiments of the invention for ease of explanation. However, it will be apparent that one or more embodiments may be practiced without these specific details. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concept of the invention.

[0032] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "comprising" as used herein indicates the presence of features, steps, or operations, but does not exclude the presence or addition of one or more other features.

[0033] When using expressions such as "at least one of A, B, and C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, and C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.). When using expressions such as "at least one of A, B, or C," the expression should generally be interpreted in accordance with the meaning commonly understood by a person skilled in the art (e.g., "a system having at least one of A, B, or C" should include, but is not limited to, systems having A alone, having B alone, having C alone, having A and B, having A and C, having B and C, and / or having A, B, and C, etc.).

[0034] In this invention, the term "Py-COOH" refers to pyridone compounds with carboxyl modification.

[0035] In this invention, the term "Py-COOH@ZIF-8" refers to a composition with Py-COOH as the core and ZIF-8, an organometallic framework material, as the shell.

[0036] In this invention, the term "Py-COOH-" 1 "O2@ZIF-8" refers to Py-COOH@ZIF-8 carrying singlet oxygen.

[0037] The term "treatment" refers to exposing a subject to (e.g., administering medication) a drug, composition, or the like based on the present invention after the subject has contracted a disease, thereby reducing the symptoms of the disease compared to when the subject has not been exposed, and does not imply the necessity of completely suppressing the symptoms of the disease. Contracting a disease means the appearance of symptoms of a disease in the body.

[0038] The term "prevention" refers to the reduction of symptoms after contracting a disease by exposing (e.g., administering medication) a subject to a drug, composition, or the like based on the present invention before the onset of the disease, compared to the absence of exposure, and does not imply the necessity of completely suppressing the disease.

[0039] In realizing the concept of this invention, it was discovered that the treatment of Helicobacter pylori in related technologies uses quadruple therapy (i.e., treatment with two antibiotics combined with a proton pump inhibitor and bismuth). The choice of antibiotics is based on the drug sensitivity results of the infected strain or on the doctor's empirical treatment; commonly used antibiotics include amoxicillin, clarithromycin, furazolidone, and metronidazole. However, this approach currently has some drawbacks, leading to treatment failure in patients. The reasons for this are considered to be as follows: 1. The drug is affected by the gastric environment; gastric acid conditions can inactivate the drug, gastric emptying reduces the interaction time between the drug and bacteria, and the mucus-bicarbonate barrier covering the gastric mucosal epithelial cells also prevents drug penetration. These influencing factors can lead to excessively low drug concentrations at bacterial growth sites, failing to completely eradicate Helicobacter pylori and increasing the possibility of recurrence. 2. Mutations in the chromosomal coding of Helicobacter pylori alter the therapeutic target of antibiotics, or impaired regulation of drug uptake and / or efflux, along with the formation of biofilms and spheroids, can lead to single-drug or multi-drug resistance, rendering conventional doses ineffective. 3. Clinical treatment for Helicobacter pylori often requires the continuous use of four medications for up to two weeks, involving high doses. The intake of antibiotics can cause intestinal dysfunction, leading to constipation or diarrhea; proton pump inhibitors can cause insufficient gastric acid secretion, resulting in indigestion; and bismuth preparations may cause nausea and vomiting. These adverse reactions can make patients unable to tolerate the medication and discontinue use on their own, significantly reducing the treatment's effectiveness.

[0040] Specifically, according to one embodiment of the invention, a composition is provided comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework.

[0041] In some specific embodiments of the present invention, acid-degradable organometallic frameworks, or acid-responsive organometallic framework materials (MOFs), are materials that can decompose under acidic conditions. They are typically formed by the self-assembly of metal ions or metal clusters with organic ligands, possessing high porosity and tunable chemical properties. Under acidic conditions, the degraded organometallic frameworks can release metal ions. In some embodiments, the acidic conditions described herein include, for example, suitable pH values ​​less than 7.0, such as pH values ​​below 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, 2.0, 1.5, 1.0, etc. In some embodiments, the acidic conditions described herein include a gastric acid environment; for example, under normal circumstances, the gastric pH value is typically maintained between 1.5 and 3.5; under special physiological conditions or disease influences, the gastric pH value may fluctuate in the range of 1.0 to 4.0. Therefore, in some embodiments, the acidic conditions preferably include gastric acidic conditions, such as pH values ​​of 1.0-4.0, for example, pH values ​​below 4.0, below 3.5, below 3.0, below 2.5, below 2.0, below 1.5, below 1.0, etc.

[0042] In some specific embodiments of the present invention, pyridone carboxyl derivatives can capture, carry, and release singlet oxygen (…). 1 O2 carries singlet oxygen through chemical bonding and can interact with singlet oxygen through specific chemical reaction mechanisms (such as 2+4 cycloaddition reactions), i.e., pyridone carboxyl derivatives and... 1 O2 reacts to form stable internal peroxides, which can then be continuously released without relying on oxygen or light. Furthermore, singlet oxygen, as a highly oxidizing reactive oxygen species (ROS), can attack bacterial cell membranes, leading to increased cell membrane permeability and thus disrupting cell integrity. Moreover, singlet oxygen can react with bacterial DNA, causing DNA strand breaks, thereby inhibiting bacterial replication and survival, thus achieving a bactericidal effect.

[0043] Specifically, the pyridone carboxyl derivative includes the structure shown in formula (II) or formula (III), preferably the structure shown in formula (II):

[0044] (II);

[0045] (III).

[0046] In formula (II) or formula (III), L is selected from H or C1~C6 saturated or unsaturated alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, vinyl, propenyl, ethynyl, propynyl, etc., and "-" indicates that it can be substituted at any position on the pyridone ring.

[0047] In some specific embodiments of the present invention, the pyridone derivatives include the structure shown in formula (I):

[0048] (I).

[0049] In some specific embodiments of the present invention, an acid-degraded organometallic framework is coated on a pyridone carboxyl derivative to form a core-shell structured composition.

[0050] Specifically, the core-shell structure design improves the stability of the composition, preventing the premature release of antimicrobial compounds such as singlet oxygen molecules before reaching the site of infection. The nanoscale core-shell structure can improve the targeting of drugs, making them easier to reach the site of infection and reducing side effects on normal tissues.

[0051] According to embodiments of the present invention, a composition comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework can serve as a carrier for singlet oxygen. Under acidic conditions, it degrades and releases metal ions with bactericidal functions, while continuously releasing singlet oxygen independent of oxygen and light. It can be used for bacterial infections in acidic, hypoxic, and light-free environments, such as Helicobacter pylori infection in the stomach. The released metal ions and singlet oxygen enter the cell, promoting the leakage of cell contents and interfering with the metabolism of bacteria such as Helicobacter pylori. The combined effect of both exhibits good antibacterial ability.

[0052] According to an embodiment of the present invention, the metal node material of the acid-degraded organometallic framework includes at least one of silver, zinc, and nickel.

[0053] According to embodiments of the present invention, silver is a metal with strong antibacterial activity. Silver ions (Ag⁺) can bind to thiol groups (-SH) on bacterial cell membranes, leading to increased cell membrane permeability and thus disrupting cell integrity. Silver ions can also bind to bacterial DNA, interfering with DNA replication and transcription, thereby inhibiting bacterial growth and reproduction. Zinc is also a metal with antibacterial activity. Zinc ions (Zn²⁺) can bind to phospholipids and proteins on bacterial cell membranes, altering cell membrane permeability and causing leakage of cell contents. Zinc ions can also bind to key bacterial enzymes, inhibiting their activity and thus interfering with bacterial metabolic processes. Nickel also has certain bactericidal activity. Nickel ions (Ni²⁺) can bind to phospholipids and proteins on bacterial cell membranes, altering the structure and function of the cell membrane. Nickel ions can also bind to bacterial DNA, interfering with DNA replication and transcription, thereby achieving an antibacterial effect.

[0054] According to embodiments of the present invention, the organometallic framework material can be at least one of Ag-MOFs, Ag / Zn-MOFs, Ni-MOF-74 and its derivatives, ZIF-8 and its derivatives, and Zn-MOF-74.

[0055] According to an embodiment of the present invention, in an acidic environment, the metal nodes silver, zinc or nickel in the MOFs material will gradually degrade, releasing the corresponding metal ions, which will synergistically achieve antibacterial effects with the carried singlet oxygen.

[0056] In some specific embodiments of the present invention, the organometallic framework material is preferably a zinc-based zeolite-imidazolium ester framework material (ZIF-8).

[0057] According to an embodiment of the present invention, ZIF-8 has a high specific surface area and a regular pore structure, which can effectively load drugs or antibacterial molecules. ZIF-8 releases Zn through cleavage in an acidic environment such as gastric acid. 2+ It can disrupt the integrity of bacterial cell membranes, promote bacterial oxidative stress and generate free radicals, and the binding with singlet oxygen can significantly enhance the antibacterial effect. Especially in the form of nanomedicines, it can improve the drug's targeting and bioavailability.

[0058] According to an embodiment of the present invention, the composition further includes singlet oxygen molecules carried therein; preferably, the composition is a nanomedicine.

[0059] According to embodiments of the present invention, the composition achieves a highly efficient antibacterial effect by releasing metal ions from MOFs through acid degradation, utilizing the antibacterial activity of pyridone carboxyl derivatives, and leveraging the strong oxidizing power of singlet oxygen. Simultaneously, the nanomedicine form enhances drug targeting and bioavailability, reduces the risk of drug resistance, and possesses potential anti-inflammatory and immunomodulatory effects.

[0060] According to another embodiment of the present invention, a method for preparing a composition comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework, and a singlet oxygen molecule carried therein is provided, exemplarily described herein. Figure 1 Py-COOH- 1 A schematic diagram of O2@ZIF-8 preparation is shown, including:

[0061] Adding a photosensitizer to a pyridone carboxyl derivative and then introducing oxygen yields a pyridone derivative carrying a singlet oxygen molecule.

[0062] The pyridone carboxyl derivatives carrying singlet oxygen molecules are combined with organometallic frameworks through adsorption to obtain a composition with a core-shell structure.

[0063] According to an embodiment of the present invention, by adding a photosensitizer to a pyridone carboxyl derivative and introducing oxygen, singlet oxygen molecules can be generated and carried rapidly and effectively. The pyridone derivative carrying the carboxyl group has a negative charge on its surface, while the organometallic framework has a positive charge on its surface. The pyridone carboxyl derivative carrying the singlet oxygen molecule and the organometallic framework combine through charge adsorption to form a composition with a core-shell structure, which can protect the singlet oxygen molecule from being easily released and at the same time provide a controllable release mechanism.

[0064] According to embodiments of the present invention, the photosensitizer includes at least one of dihydroporphyrin E6, methylene blue, or indocyanine green.

[0065] According to embodiments of the present invention, under light conditions, dihydroporphyrin E6, methylene blue, or indocyanine green can all efficiently convert oxygen into singlet oxygen molecules. One of them can be selected or used in combination. These photosensitizers can efficiently convert oxygen into singlet oxygen molecules under light conditions, providing highly reactive oxygen species for subsequent applications.

[0066] According to another aspect of the present invention, a composition comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework is provided for use in the preparation of a delivery carrier or medicament for the treatment / prevention of Helicobacter pylori infection.

[0067] According to embodiments of the present invention, a composition comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework can be used to prepare a delivery carrier or drug for treating / preventing Helicobacter pylori infection. This carrier carries antibacterial active substances such as singlet oxygen. In the acidic environment of the stomach, the acid-degraded organometallic framework cleaves upon contact with acid, releasing singlet oxygen in approximately 30 minutes. The synergistically cleaved metal ions remove Helicobacter pylori, ensuring that the antibacterial process is completed while the composition remains in the stomach without affecting the intestinal flora.

[0068] Furthermore, it adopted 1 The way O2 is delivered makes 1 O2 no longer requires oxygen and light at the site of action. Furthermore, encapsulating it in an organometallic framework such as ZIF-8 enhances its effectiveness, allowing the drug to achieve a 99% antibacterial rate at a concentration of 2 μmol / mL. Moreover, fecal samples collected from mice after in vivo antibacterial treatment and 16sRNA sequencing revealed no significant difference in the abundance of gut microbiota between the mice and healthy mice.

[0069] According to another aspect of the present invention, a pharmaceutical composition is provided comprising a pyridone carboxyl derivative and an acid-degraded organometallic framework, a singlet oxygen molecule carried therein, and a pharmaceutically acceptable carrier thereof.

[0070] According to embodiments of the present invention, when the drug is an injectable formulation, a pharmaceutically acceptable carrier includes at least one of a preservative, a solvent, and a stabilizer; when the drug is a topical formulation, a pharmaceutically acceptable carrier includes at least one of a matrix, a diluent, a lubricant, and a preservative; when the drug is an oral dosage form, a pharmaceutically acceptable carrier includes at least one of a filler, a diluent, a binder, a disintegrant, a lubricant, a coating material, a flavoring agent, and a pH adjuster.

[0071] According to an embodiment of the present invention, the pharmaceutical composition is in a dosage form suitable for oral administration.

[0072] According to embodiments of the present invention, the oral dosage form can be tablets, capsules, granules, oral liquids, or suspensions, which can improve the convenience of medication for patients. Compared with traditional quadruple therapy, this dosage form can reduce the frequency and complexity of medication, thereby improving patient compliance. Moreover, Helicobacter pylori mainly colonizes the gastric mucosa surface, and the oral medication can act directly on the stomach, thereby more effectively killing the bacteria. The oral medication can achieve a high concentration in the stomach, and the acid-degraded organometallic framework can directly degrade and release singlet oxygen and metal ions under the acidic conditions of the stomach after oral administration, achieving an antibacterial effect.

[0073] The present invention will be further explained in conjunction with specific embodiments below. Unless otherwise stated, all reagents used in the following embodiments are commercially available reagents.

[0074] Example 1: Preparation of Py-COOH@ZIF-8 composition

[0075] 160 mg of 2-hydroxypyridine and 80 mg of sodium hydride were dissolved in 10 mL of anhydrous N,N-dimethylformamide and stirred at 0 °C for 1 hour. Then 84.5 μL of methyl 3-bromopropionate was added, and the reaction was continued at room temperature for 2 hours.

[0076] After the reaction was complete, the reaction solution was added dropwise to stirred ice water, and then extracted with dichloromethane. The dichloromethane phase was retained, washed with saturated brine, dried over anhydrous Na₂SO₄, and the solvent was removed by rotary evaporation to obtain the crude product. The crude product was subjected to column chromatography (methanol:dichloromethane = 1:10) to separate 2-hydroxypyridinone methyl ester. The product was dissolved in an ethanol / deionized water solution (ethanol:deionized water = 1:1) at 0 °C, and lithium hydroxide (30 mg) was added. The mixture was then transferred to 40 °C and the reaction was continued for 4 hours. After the reaction was complete, the ethanol was removed by rotary evaporation, and 300 μL of 50% hydrochloric acid solution was added to the remaining liquid to further remove deionized water, yielding the crude Py-COOH product. This product was purified by column chromatography (methanol:dichloromethane = 1:3). The solvent was removed by rotary evaporation, and the product was dried to obtain Py-COOH.

[0077] 1.8 g of zinc nitrate hexahydrate and 3.96 g of 2-methylimidazole were dissolved separately in methanol. The two solutions were then rapidly mixed and stirred at room temperature for 1 hour to obtain a white powdery solid. ZIF-8 was then purified by centrifugation.

[0078] Py-COOH carries a negative charge and can combine with positively charged ZIF-8 through charge adsorption, thus embedding Py-COOH within the pores of ZIF-8. Py-COOH and ZIF-8 are mixed in deionized water at a 1:1 mass ratio and reacted at room temperature for 6 hours to obtain an aqueous solution of Py-COOH@ZIF-8.

[0079] The prepared Py-COOH@ZIF-8 was characterized, and the results are shown in Figure 2.

[0080] Figure 2 The figures shown are the characterization results of Py-COOH@ZIF-8 in the embodiments of the present invention. A is a transmission electron microscope image of Py-COOH@ZIF-8 under neutral conditions, B is a transmission electron microscope image of Py-COOH@ZIF-8 under acidic conditions, and C is a zeta potential diagram.

[0081] according to Figure 2It can be seen that a composition with a core-shell structure, consisting of an organometallic framework as the outer shell and a pyridone carboxyl derivative as the core, was effectively prepared. It is stable under neutral conditions but will decompose under acidic conditions. Moreover, the pyridone carboxyl derivative has a negative charge on its surface and the organometallic framework has a positive charge on its surface, which can effectively bind to each other through charge.

[0082] Example 2 Py-COOH- 1 Preparation of O2@ZIF-8

[0083] Preparation process as follows Figure 1 As shown, dihydroporphyrin (Ce6) was added to an aqueous solution of Py-COOH@ZIF-8 and mixed. Oxygen was then introduced, and the mixture was irradiated with a simulated light source at 660 nm for 30 minutes to generate enough singlet oxygen to be captured, forming Py-COOH- 1 O2@ZIF-8.

[0084] Example 3 Py-COOH- 1 Characterization of singlet oxygen loading and release capacity of O2@ZIF-8

[0085] 1,3-Diphenylisobenzofuran (DPBF) was used as a probe to measure 1 O2 generation:

[0086] Dissolve Py-COOH@ZIF-8 (4 mg / mL) and Ce6 in water or PBS solution, then place the sample in an ice bath under a simulated light source (650 ± 10 nm, 80 mW / cm²). 2 The sample was irradiated for 30 minutes while oxygen was introduced into the solution. Then it was mixed with DPBF (200 µg / mL), and the curve of the mixed solution was recorded at a wavelength of 420 nm using a UV-Vis spectrometer.

[0087] Singlet oxygen (O2) in the absence of light was detected by electron spin resonance (ESR) spectroscopy. 1 O2 generation:

[0088] 2,2,6,6-Tetramethylpiperidine (TEMP) is used as 1 O2 scavenging agent. Py-COOH@ZIF-8 and Ce6 were dissolved in PBS buffer solution and subjected to simulated sunlight (650±10nm, 80mW / cm²). 2 Irradiate the solution for 30 minutes, then introduce oxygen into the solution, followed by the addition of an equal volume of TEMP singlet oxygen scavenger, and finally analyze the solution using an instrument. 1 O2 release curve.

[0089] Figure 3 The Py-COOH- of this embodiment of the invention 1UV curves of loading and releasing singlet oxygen in O2@ZIF-8; Figure 4 The Py-COOH- of this embodiment of the invention 1 Electron spin resonance spectrum of loaded and released singlet oxygen in O2@ZIF-8.

[0090] according to Figure 3 and Figure 4 It can be seen that, under the excitation conditions free from singlet oxygen, Py-COOH- 1 O2@ZIF-8 can still stably release singlet oxygen.

[0091] Example 4 Py-COOH- 1 Validation of the in vitro antibacterial activity of O2@ZIF-8

[0092] After removing the frozen Helicobacter pylori from the -80°C freezer, it was thawed in a 37°C water bath. Then, it was evenly spread onto Columbia agar medium containing 5% sheep blood and incubated for 48 hours at 37°C, 5% O2, and 10% CO2. Once the bacteria had confluently grown in the medium, a suitable amount of bacteria was scraped off using a disposable spreader and dispersed in Columbia broth medium (pre-adjusted for pH with dilute hydrochloric acid), adjusting the density to 1×10⁻⁶. 8 CFU / mL.

[0093] Different concentrations of Py-COOH@ZIF-8 and Ce6 were dissolved in water or PBS solution, and then the samples were placed in an ice bath under a simulated light source (650±10nm, 80mW / cm²). 2 The sample was irradiated for 30 minutes while oxygen was simultaneously bubbled into the solution to prepare Py-COOH- 1 O2@ZIF-8. Take 180µL Py-COOH- 1 O2@ZIF-8 and 20 µL of bacterial culture were incubated in 96-well plates at 37°C in a 5% O2, 10% CO2 incubator for 24 h. The OD value was then measured at 660 nm using a microplate reader, and the inhibition rate was calculated. The results are as follows: Figure 5 As shown.

[0094] Figure 5 This invention relates to Py-COOH- 1 Figure showing the in vitro antibacterial and bacteriostatic rate results of O2@ZIF-8.

[0095] according to Figure 5 It can be seen that in an environment with pH=3, Py-COOH- 1 O2@ZIF-8 can achieve an antibacterial rate of over 99% at a concentration of 2 μmol / mL, and its antibacterial effect is better under acidic conditions than under neutral conditions.

[0096] Example 5 Py-COOH- 1 In vitro antibacterial activity characterization of O2@ZIF-8

[0097] Different concentrations of Py-COOH@ZIF-8 and Ce6 were dissolved in water or PBS solution, and then the samples were placed in an ice bath under a simulated light source (650±10nm, 80mW / cm²). 2 The sample was irradiated for 30 minutes while oxygen was simultaneously bubbled into the solution to prepare Py-COOH- 1 O2@ZIF-8. Take bacterial suspension (density 1×10⁻⁶). 8 After thoroughly mixing and co-culturing with the Py-COOH solution (CFU / mL), the culture medium was washed away three times by centrifugation. The resulting precipitate was then fixed by pipetting and mixing with 2.5% glutaraldehyde solution for 4 hours. Subsequently, it was serially diluted with 25%, 50%, and 75% ethanol, and finally dispersed in anhydrous ethanol. A drop of the bacterial suspension was placed on a coverslip, allowed to dry, and then sputter-coated with gold. The precipitate was then observed under a scanning electron microscope (SEM). 1 Bacterial morphology after O2@ZIF-8 treatment, results are as follows Figure 6 As shown.

[0098] Figure 6 This invention relates to Py-COOH- 1 Image of O2@ZIF-8 in vitro antibacterial plating plate and scanning electron microscopy characterization results.

[0099] according to Figure 6 It can be seen that after Py-COOH- 1 The bacteria treated with O2@ZIF-8 showed obvious rupture and collapse in morphology, while the bacteria in the control group were plump and grew well.

[0100] Example 6 Py-COOH- 1 O2@ZIF-8 antibacterial activity in vivo and animal experiments

[0101] (1) Constructing a mouse model of Helicobacter pylori infection:

[0102] Thirty 5-week-old Balb / c mice were purchased and randomly divided into 6 groups (healthy mouse group, PBS group, Py-COOH group, Py-COOH@ZIF-8 group, Py-COOH-...). 1 (O2@ZIF-8 group and antibiotic group), 5 mice in each group. Mice in each group were first administered NaHCO3 by gavage, and 0.3 mL of 1×10⁻⁶ antibiotics one hour later. 8 CFU / mL bacterial solution was administered by gavage once daily for four consecutive days.

[0103] (2) Identification of Helicobacter pylori infection mouse model

[0104] Four weeks after Helicobacter pylori infection, two mice were randomly sacrificed, and their stomachs were removed. A portion of the gastric tissue was homogenized, diluted, and spread onto Columbia agar plates containing 5% sheep blood. The plates were incubated at 37°C, 5% O2, and 10% CO2 for 48 hours. Bacterial growth was then observed. A portion of fresh gastric mucosa was taken and placed in urea solution, then phenolphthalein was added to observe the color change. The remaining tissue was dehydrated, embedded in paraffin, and sectioned for HE staining to observe the degree of gastric mucosal inflammation.

[0105] (3) Antibacterial experiment in mice

[0106] After confirming bacterial colonization, mice were fasted for 12 hours and then administered 0.5 mL of the drug via gavage for 4 consecutive days, during which feces were collected. On the day after the last gavage, some mice were sacrificed, and their stomachs were removed. A portion of the stomach tissue was homogenized, diluted, and spread onto Columbia agar plates containing 5% sheep blood. The mixture was incubated at 37°C, 5% O2, and 10% CO2 for 48 hours. Bacterial growth was then observed. The remaining tissue was dehydrated, embedded in paraffin, and stained with hematoxylin and eosin (HE) to observe the gastric mucosal inflammation. The results are as follows: Figure 7 As shown.

[0107] Figure 7 This is a diagram showing the HE staining results of an in vivo antibacterial experiment in mice according to an embodiment of the present invention.

[0108] according to Figure 7 It can be seen that gastric inflammation still existed in the mice in the PBS control group, but after Py-COOH- 1 The inflammation in the stomachs of mice treated with O2@ZIF-8 via gavage has significantly subsided, indicating that light stimulation produces... 1 Py-COOH- formed by O2 1 O2@ZIF-8 has a good effect on eliminating Helicobacter pylori colonizing the stomach.

[0109] Example 7 Py-COOH- 1 Validation of O2@ZIF-8 biosafety

[0110] (1) Hemolysis test

[0111] Different concentrations (0, 1, 2, 4, 8 μmol / mL) of Py-COOH@ZIF-8 and Ce6 were dissolved in water or PBS solution. The samples were then placed in an ice bath under a simulated light source (650±10 nm, 80 mW / cm²). 2 The sample was irradiated for 30 minutes while oxygen was simultaneously bubbled into the solution to prepare Py-COOH- 1O2@ZIF-8.

[0112] Fresh blood was centrifuged at 2500 rpm, the supernatant was carefully aspirated, and red blood cells were retained. PBS was then added, and the process was repeated several times. Next, 500 μL of PBS solution containing 10% RBCs and 500 μL of Py-COOH- were added. 1 O2@ZIF-8 was prepared into a 1 mL solution. Simultaneously, 50 μL of red blood cells were added to PBS to prepare a 1 mL negative control, and 50 μL of red blood cells were added to deionized water to prepare a 1 mL positive control. After incubation at 37°C for 2 hours, the mixture was centrifuged, and the supernatant was transferred to a 96-well plate. The absorbance of the supernatant at 540 nm was measured using a microplate reader. The hemolysis rate was calculated, and the results are as follows: Figure 8 As shown:

[0113] Hemolysis rate (%) = (OD) 实验组 –OD 阴性对照组 ) / (OD 阳性对照组 –OD 阴性对照组 )×100%,

[0114] Figure 8 This invention relates to Py-COOH- 1 Image of the results of the O2@ZIF-8 hemolysis experiment.

[0115] according to Figure 8 It can be seen that at the antibacterial concentration, Py-COOH- 1 The presence of O2@ZIF-8 almost never causes hemolysis.

[0116] (2) Cell compatibility test

[0117] Different concentrations (0, 1, 2, 4, 8 μmol / mL) of Py-COOH@ZIF-8 and Ce6 were dissolved in water or PBS solution. The samples were then placed in an ice bath under a simulated light source (650±10 nm, 80 mW / cm²). 2 The sample was irradiated for 30 minutes while oxygen was simultaneously bubbled into the solution to prepare Py-COOH- 1 O2@ZIF-8.

[0118] Gastric mucosal epithelial cells (GES-1) were cultured in medium containing 10% fetal bovine serum (FBS) at 37°C and 5% CO2 for 24 hours. They were then seeded at a density of 2000 cells per well in 96-well plates until the cells reached approximately 80% confluence. The medium was then removed, and 100 μL of a solution containing different concentrations of Py-COOH- was added. 1The cells were co-incubated with O2@ZIF-8 complete culture medium for 1 day. The supernatant was then aspirated, and 100 μL of 0.5% MTT was added, followed by incubation for another 4 hours. The supernatant was then removed, and 150 μL of LDMSO was added to each well to dissolve the cells. The absorbance of each well was measured at 490 nm using a microplate reader, and cell viability was calculated. The results are shown below. Figure 9 As shown:

[0119] Cell viability (%) = (OD) 实验组 –OD 空白对照组 ) / (OD PBS组 –OD 空白对照组 )×100%.

[0120] Figure 9 This invention relates to Py-COOH- 1 Figure showing the results of the O2@ZIF-8 cell compatibility experiment.

[0121] according to Figure 9 It can be seen that, in terms of effective antibacterial concentration, Py-COOH- 1 After co-culturing O2@ZIF-8 and GES-1 for 24 hours, the cell viability remained above 95%.

[0122] (3) Mouse histological staining

[0123] The day after the last gavage, some mice were sacrificed. Organ specimens (heart, liver, spleen, lung, kidney) were fixed, dehydrated, and embedded in paraffin. Hematoxylin and eosin (H&E) staining was used for observation. The results are as follows: Figure 10 As shown.

[0124] Figure 10 This invention relates to Py-COOH- 1 O2@ZIF-8 biosafety histological staining results.

[0125] according to Figure 10 It can be seen that no obvious histological abnormalities or inflammatory lesions were observed in the major organs after treatment.

[0126] (4) Serological examination of mice

[0127] Some mice were sacrificed the day after the last gavage, and blood was collected from their eyeballs for blood cell counts and biochemical examinations. The results showed that the treatment had no adverse effects on liver and kidney function or blood cell counts in the mice.

[0128] (5) Analysis of mouse gut microbiota

[0129] Fresh fecal samples were collected from mice. The abundance and diversity of bacteria in mouse feces, as well as the relative abundance of colony structures, were determined by Lingen Biotechnology Co., Ltd. (Shanghai) using 16S rRNA sequencing. The results are as follows: Figure 11 As shown.

[0130] Figure 11 This invention relates to Py-COOH- 1 The results of the O2@ZIF-8 biosafety mouse gut microbiota analysis are shown in Figure A, which is a bar chart of the abundance of mouse fecal microbiota; Figure B is a heatmap of the abundance of mouse fecal microbiota; and Figure C is a graph showing the results of the functional prediction analysis of mouse gut microbiota.

[0131] according to Figure 11 It can be seen that the sequencing results indicate that, for the analysis of the species composition of feces, Py-COOH- 1 The fecal microbiota of mice treated with O2@ZIF-8 was more similar to that of healthy mice, Py-COOH- 1 O2@ZIF-8 treatment has a smaller impact on fecal microbiota, which reflects the Py-COOH- of this invention. 1 O2@ZIF-8 has excellent biocompatibility.

[0132] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A composition for preparing a delivery carrier or drug for treating / preventing Helicobacter pylori infection, comprising a singlet oxygen molecule, a pyridone carboxyl derivative, and an acid-degraded organometallic framework carried therein; in, The acid-degraded organometallic framework is a zinc-based zeolite-imidazolium ester framework material; The pyridone carboxyl derivative comprises the structure shown in formula (I): （I）； as well as The preparation method of the composition includes: adding a photosensitizer to a pyridone carboxyl derivative and introducing oxygen to obtain a pyridone derivative carrying a singlet oxygen molecule; The pyridone carboxyl derivative carrying singlet oxygen molecules is combined with an organometallic framework through adsorption to obtain a composition with a core-shell structure.

2. The composition of claim 1, wherein the composition is a nanomedicine.

3. The composition according to claim 1, wherein, The photosensitizer includes at least one of dihydroporphyrin E6, methylene blue, or indocyanine green.

4. The use of the composition according to any one of claims 1 to 3 in the preparation of a delivery carrier or medicament for the treatment / prevention of Helicobacter pylori infection.

5. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier thereof.

6. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is a dosage form suitable for oral administration.

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