Acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres, and preparation method and application thereof

Abstract

The embodiment of the application discloses an acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microsphere and a preparation method and application thereof. The microsphere comprises an oxygen-releasing inner core taking polycaprolactone and calcium peroxide as base materials, and a pH-responsive outer shell modified by chitosan through N-acetyl-d-glucosamine and loaded with a natural medicine 6-Gin; the microsphere is disintegrated in the outer shell under the gastric acid environment and rapidly releases 6-Gin, and the inner core continuously releases oxygen by reacting with water. The microsphere provided by the application can effectively inhibit bacterial adhesion, destroy a biofilm and directly kill H. pylori, and solves the problem of poor curative effect caused by a microenvironment in traditional therapy, and provides a new effective strategy for anti-H. pylori therapy.

Description

Technical Field

[0001] This invention relates to the field of drug delivery technology, specifically to an acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microsphere, its preparation method, and its application. Background Technology

[0002] Helicobacter pylori (H. pylori) infection is a significant risk factor for chronic gastritis, peptic ulcers, and even gastric cancer, and its eradication remains a clinical challenge. Currently, standard clinical therapies centered on antibiotics are not only prone to bacterial resistance but also frequently lead to secondary resistance and infection recurrence due to systemic administration, as well as serious side effects such as gastrointestinal discomfort and dysbiosis. Therefore, developing novel drug delivery systems that can target the stomach, act locally, and overcome bacterial resistance mechanisms is crucial.

[0003] Gastric mucosal adhesive microspheres, as a local delivery strategy, can prolong the residence time of formulations in the stomach, thereby increasing drug concentration at the site of infection. Among these, chitosan, a natural cationic polymer, is widely used to construct such delivery systems due to its excellent biocompatibility, biodegradability, and mucosal adhesion. Existing technologies include studies using chitosan microspheres loaded with drugs for anti-H. pylori treatment. However, these systems have relatively limited functionality, with most active ingredients being chemical drugs, and face challenges in loading and controlling the release of natural drugs. More importantly, H. pylori can form a dense biofilm on the gastric mucosa, creating a microaerophilic microenvironment for the strain's survival. This dense biofilm severely weakens the antibacterial efficacy of most drugs, a key factor leading to treatment failure, a bottleneck that traditional adhesive microsphere technology struggles to effectively address.

[0004] Calcium peroxide (CaO2), as a potential oxygen-releasing carrier, has been extensively studied for tissue oxygenation. Polycaprolactone (PCL), a hydrophobic and biodegradable polyester, while hydrophobic in structure, still allows for slow water molecule permeation, thus making it suitable for encapsulating CaO2. By controlling the water molecule permeation rate, the oxygen release time can be prolonged, constructing a long-lasting oxygen release system. However, such oxygen release systems typically lack intelligent responsiveness to the acidic environment of the stomach and are not synergistically integrated with effective therapeutic drugs, making it difficult to achieve precise and synergistic therapeutic effects on H. pylori infection lesions.

[0005] In summary, due to the microaerophilic microenvironment of the *H. pylori* biofilm and the complex pH conditions in the stomach, existing single-therapies or simple delivery systems (such as simple adhesive microspheres or oxygen-releasing materials) struggle to achieve on-demand, synergistic release of therapeutic drugs and oxygen. Therefore, integrating intelligent oxygen-releasing systems with pH-responsive drug controlled-release and highly effective natural antibacterial drugs to construct a synergistic therapeutic system capable of simultaneously disrupting the microaerophilic microenvironment of the biofilm and enhancing bactericidal efficacy is a pressing technical challenge that needs to be addressed. Summary of the Invention

[0006] Therefore, this invention provides an acid-responsive oxygen-releasing chitosan / polycaprolactone (PCL) core-shell microsphere, its preparation method, and its applications. These microspheres can intelligently and synergistically release the antibacterial drug 6-gingerol (6-Gin) and oxygen in the acidic environment of the stomach, significantly inhibiting H. pylori adhesion, disrupting its biofilm structure, and adjusting the microaerophilic state within the biofilm. They exhibit strong growth-inhibiting activity against H. pylori, with low cytotoxicity and good biosafety.

[0007] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0008] According to a first aspect of the present invention, the present invention provides an acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microsphere, comprising an oxygen-releasing core based on polycaprolactone and calcium peroxide, and a pH-responsive shell made of chitosan modified with N-acetyl-d-glucosamine (GlcNAc) and loaded with the natural drug 6-Gin; the microspheres disintegrate in the acidic environment of the stomach and rapidly release 6-Gin, while the core reacts with water to continuously release oxygen.

[0009] Furthermore, the microspheres are regular spherical in shape, with a diameter of 20-30 μm, a core diameter of 15-20 μm, and a shell thickness of 5-10 μm.

[0010] Furthermore, in a release medium with pH=2.0, the microspheres exhibit a cumulative release rate of 6-Gin greater than 90% within 24 hours and a continuous oxygen release time exceeding 72 hours; in a release medium with pH=7.4, the cumulative release rate of 6-Gin is less than 30% within 24 hours.

[0011] According to a second aspect of the present invention, the present invention provides a method for preparing acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres as described above, comprising the following steps:

[0012] (1) Core fluid preparation: Calcium peroxide was ultrasonically dispersed in a dichloromethane solution of polycaprolactone, filtered, and a homogeneous core fluid was obtained.

[0013] (2) Preparation of shell fluid: Chitosan was dissolved in an aqueous acetic acid solution, N-acetyl-d-glucosamine and drug 6-Gin were added, and the mixture was stirred until completely dissolved to obtain shell fluid;

[0014] (3) Microsphere preparation and curing: Using a coaxial electrostatic spraying device, the core fluid obtained in step (1) and the outer shell fluid obtained in step (2) are simultaneously pumped out through the inner and outer needles of the coaxial nozzle, respectively, and sprayed into the sodium tripolyphosphate solution under a high voltage electrostatic field for cross-linking and curing. After collection, washing and drying, the microspheres are obtained.

[0015] Further, in step (1), the mass concentration of calcium peroxide in the core fluid is 8%-12%, and the mass concentration of polycaprolactone is 6%-10%.

[0016] Further, in step (2), the mass fraction of the acetic acid aqueous solution is 0.6-1.2%, the mass concentration of chitosan in the shell fluid is 1.5%-2.5%, the mass concentration of N-acetyl-d-glucosamine is 8%-12%, and the mass concentration of 6-Gin is 0.4%-0.6%.

[0017] Furthermore, the parameters of the coaxial electrostatic spray are as follows: voltage 10-15 kV, core fluid flow rate 0.3-0.5 mL / h, outer fluid flow rate 1.5-2.5 mL / h, receiving distance 12-18 cm, inner needle inner diameter 0.15-0.2 mm, outer diameter 0.3-0.35 mm, outer needle inner diameter 0.6-0.65 mm, outer diameter 1.1-1.15 mm.

[0018] Furthermore, in step (3), the mass concentration of the sodium tripolyphosphate solution is 0.8%-1.2%;

[0019] The mass concentration mentioned in this invention refers to the mass-volume concentration, in g / mL; the mass fraction refers to the percentage of solute mass to the total mass of the solution.

[0020] The conditions for crosslinking and curing are: room temperature, 5-15 min;

[0021] The drying conditions were: pre-freezing at -80℃ for 24 h, followed by vacuum freeze-drying for 48 h.

[0022] According to a third aspect of the present invention, the present invention provides the use of acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres as described above, or acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres prepared by any of the methods described above, in the preparation of drugs or foods for treating Helicobacter pylori or relieving gastritis.

[0023] Furthermore, the acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres can inhibit Helicobacter pylori adhesion, disrupt its biofilm structure, alleviate the hypoxic microenvironment inside the biofilm, and exhibit growth-inhibiting activity against Helicobacter pylori.

[0024] The core-shell microspheres provided by this invention are constructed using coaxial electrospray technology. The core fluid consists of calcium peroxide (CaO2) encapsulated in polycaprolactone (PCL), while the outer shell fluid is N-acetyl-d-glucosamine (GlcNAc)-modified chitosan loaded with the natural drug 6-gingerol (6-Gin). In the outer shell layer, the GlcNAc-modified chitosan utilizes its cationic properties to electrostatically adsorb onto the negatively charged gastric mucosa, achieving long-term retention of the microspheres in the stomach. Simultaneously, this modification endows the material with acid-responsive drug release characteristics—in the acidic environment of the stomach, the protonation degree of chitosan increases, its structure becomes loose and dissolves, thereby triggering the release of the loaded 6-Gin at the site of infection, exerting antibacterial and anti-inflammatory effects. In the core layer, PCL acts as a hydrophobic matrix to encapsulate CaO2. Its moderate hydrophobicity slows down the inward penetration of water molecules, allowing the reaction between CaO2 and water (2CaO2 + 2H2O → 2Ca(OH)2 + O2↑) to proceed slowly and continuously. This enables the long-term release of oxygen, thereby improving the hypoxic state of the gastric infection microenvironment and synergistically enhancing the antibacterial effect. To obtain ideal microspheres with a complete core-shell structure, suitable core-shell thickness, and high drug loading, precise control of multiple parameters is required: the core fluid must ensure uniform dispersion of CaO2 in the PCL solution and possess suitable viscosity to guarantee stable spraying; the outer shell fluid consists of chitosan, GlcNAc, and the small molecule drug 6-Gin, where chitosan, as the shell framework material, determines the film-forming properties and mechanical strength of the microspheres; the incorporation of GlcNAc provides microsphere targeting and enhances adhesion; the small molecule drug 6-Gin does not participate in shell formation, but excessive concentration will damage the shell structure, therefore, each component must be controlled within a suitable range, while ensuring the system has suitable conductivity to form a stable coating layer; in the electrospray process, the flow rates of the inner and outer layers and their ratio are key parameters for controlling the core-shell thickness and ensuring complete coating, while the applied voltage, receiving distance, and solvent system jointly determine the uniformity of the microsphere morphology and the clear structure. Through the synergistic control of the above-mentioned material design and process parameters, the microsphere system provided by this invention has a complete core-shell structure, suitable core-shell thickness and high drug loading capacity. It integrates mucosal adhesion, acid-responsive drug release and long-term oxygen supply, significantly prolonging the drug retention time in the stomach, increasing the local drug concentration and improving the infection microenvironment, thereby overcoming the problems of frequent dosage, large systemic side effects and low eradication rate of traditional therapies.

[0025] The embodiments of the present invention have the following advantages:

[0026] The core-shell microspheres provided by this invention achieve synergistic and intelligent release of drugs and oxygen in the acidic environment of the stomach through a unique core-shell structure design and material selection. Specifically, the GlcNAc-chitosan in the outer shell provides pH responsiveness, enabling targeted and rapid drug release; the PCL / CaO2 in the inner core controls the continuous release of oxygen, effectively improving the microaerophilicity of lesions. The synergistic effect of these two components effectively inhibits bacterial adhesion, disrupts biofilms, and directly kills *H. pylori*, solving the problem of poor efficacy due to the microenvironment in traditional therapies and providing a new and effective strategy for anti-*H. pylori* treatment. Attached Figure Description

[0027] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0028] Figure 1 This is a schematic diagram of the preparation process of 6-Gin@GlcNAc@CaO2 core-shell microspheres provided in Example 1 of the present invention.

[0029] Figure 2 The image shows the physical images of the 6-Gin and 6-Gin@GlcNAc@CaO2 core-shell microspheres provided in Embodiment 1 of the present invention.

[0030] Figure 3 The optical microscope characterization image of 6-Gin@GlcNAc@CaO2 provided in Embodiment 1 of the present invention.

[0031] Figure 4 The image is a scanning electron microscope (SEM) image of 6-Gin@GlcNAc@CaO2 provided in Embodiment 1 of the present invention.

[0032] Figure 5 The image shows a fluorescence micrograph of 6-Gin@GlcNAc@CaO2 provided in Example 1 of this invention, in which the red fluorescent label is for the outer shell (chitosan) and the green fluorescent label is for the core (PCL / CaO2).

[0033] Figure 6 This is an optical microscope characterization image of 6-Gin@GlcNAc@CaO2 provided in Comparative Example 1 of the present invention.

[0034] Figure 7 This is an optical microscope characterization image of 6-Gin@GlcNAc@CaO2 provided in Comparative Example 2 of the present invention.

[0035] Figure 8The results show the effect of 6-Gin concentration on drug loading of microspheres provided by this invention.

[0036] Figure 9 An optical microscope characterization image of the microspheres prepared when the feed concentration is increased to 1% according to the present invention.

[0037] Figure 10 The in vitro drug cumulative release curves of 6-Gin@GlcNAc@CaO2 under different pH conditions provided in Example 1 of the present invention are shown.

[0038] Figure 11 The oxygen release kinetic curves of 6-Gin@GlcNAc@CaO2 under different pH conditions provided in Example 1 of the present invention.

[0039] Figure 12 The image shows the viability test results of GES-1 cells after being treated with 6-Gin@GlcNAc@CaO2 microspheres at different concentrations, as provided in Example 1 of this invention.

[0040] Figure 13 This is a statistical graph showing the effect of different concentrations of 6-Gin@GlcNAc@CaO2 microspheres provided in Example 1 of the present invention on the in vitro adhesion rate of H. pylori.

[0041] Figure 14 This is an immunofluorescence staining image of a hypoxia probe, showing the alleviating effect of different concentrations of microspheres on the microaerophilic state within the H. pylori biofilm.

[0042] Figure 15 The graph shows the statistical activity of 6-Gin@GlcNAc@CaO2 microspheres provided in Example 1 of this invention against the in vitro growth inhibition of H. pylori at different concentrations.

[0043] Figure 16 A statistical graph for flow cytometry analysis of the microsphere uptake mechanism of H. pylori.

[0044] Figure 17 Fluorescent images of H. pylori uptake behavior of microspheres at different time points observed by confocal laser scanning microscopy.

[0045] Figure 18 The graph shows the results of the determination of the minimum inhibitory concentration (MIC) of 6-Gin@GlcNAc@CaO2 microspheres, free 6-Gin, and amoxicillin against H. pylori provided in Example 1 of this invention. Detailed Implementation

[0046] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0047] Unless otherwise specified, all raw materials used in this invention are commercially available or commonly used in the field; unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0048] Example 1: Preparation of 6-Gin@GlcNAc@CaO2 core-shell microspheres (with appendix) Figure 1 )

[0049] (1) Core fluid preparation: Dissolve 90 mg PCL in 1 mL dichloromethane to prepare a 9% PCL solution, and filter it through a 0.45 μm filter membrane; weigh 10 mg CaO2 powder and add it to the above PCL solution, sonicate for 30 minutes to make it fully dispersed and uniform, and avoid agglomeration, to obtain a PCL / DCM core fluid containing 10% CaO2.

[0050] (2) Preparation of the outer shell fluid: Dissolve 20 mg of chitosan in a container containing 1 mL of 1% acetic acid aqueous solution, and stir magnetically until completely dissolved to obtain a 2% chitosan solution; add 10 mg of GlcNAc and 0.5 mg of 6-Gin to this solution, and continue stirring until all components are completely dissolved and mixed evenly;

[0051] (3) Microsphere preparation and curing: A coaxial electrostatic spraying device with two independent fluid channels (inner and outer layers) was used to inject the core fluid prepared in step (1) and the shell fluid prepared in step (2) into two syringes and fix them on the injection pump. The flow rate of the core fluid was set to 0.4 mL / h and the flow rate of the shell fluid was set to 2 mL / h. A high voltage electrostatic discharge of 12 kV was applied and the receiving distance was set to 15 cm. The core-shell microspheres formed by spraying were directly collected in a 1% TPP aqueous solution under magnetic stirring and crosslinked and cured at room temperature (25℃) for 10 minutes. Then, the microspheres were collected by centrifugation at 3000 rpm for 5 minutes, washed 3 times with PBS buffer to remove residual TPP, and finally freeze-dried to obtain light yellow 6-Gin@GlcNAc@CaO2 powder (see attached image). Figure 2 ).

[0052] Comparative Example 1: Preparation of Microspheres

[0053] The difference between the preparation of the microspheres provided in this comparative example and that in Example 1 is that the concentration of the PCL solution in step (1) is 12%. Specifically, 120 mg of PCL is dissolved in 1 mL of dichloromethane to prepare a 12% PCL solution, which is then filtered through a 0.45 μm filter membrane. 10 mg of CaO2 powder is weighed and added to the above PCL solution, and ultrasonically treated for 30 minutes to ensure that it is fully dispersed and uniform, avoiding agglomeration, thus obtaining the core fluid.

[0054] Comparative Example 2: Preparation of Microspheres

[0055] The difference between the preparation of the microspheres provided in this comparative example and that in Example 1 is that in step (2), the concentration of the chitosan solution is 1%. Specifically, 10 mg of chitosan is dissolved in a container containing 1 mL of 1% acetic acid aqueous solution and magnetically stirred until completely dissolved to obtain a 1% chitosan solution. 10 mg of GlcNAc and 0.5 mg of 6-Gin are added to this solution and stirring is continued until all components are completely dissolved and mixed evenly.

[0056] Test Example 1: Characterization of 6-Gin@GlcNAc@CaO2

[0057] The morphology and structure of the 6-Gin@GlcNAc@CaO2 prepared in Example 1 were characterized. Optical microscopy revealed that the microspheres were regularly spherical, uniform in size, and distributed between 20-30 μm in diameter. The core diameter was 15-17 μm, and the shell thickness was 4-6 μm, with a clearly visible core-shell boundary (see attached image). Figure 3 Scanning electron microscopy revealed that the microspheres had smooth surfaces and intact sphericity. Further analysis of the internal structure by breaking up some microspheres showed that they possessed a distinct layered structure of a core and outer shell. (See attached image) Figure 4 Fluorescence microscopy characterization results showed that the outer layer of the microspheres exhibited a red fluorescent ring, while the interior was filled with green fluorescence. The area where the two overlapped showed a yellow color, clearly demonstrating that it possesses a complete core-shell structure. (See attached image) Figure 5 ).

[0058] Comparative Example 1 microspheres, observed under an optical microscope, exhibit a core-shell structure, but the core volume accounts for over 80% of the total microsphere volume, while the outer shell is extremely thin (see attached image). Figure 6 Furthermore, due to the high concentration of PCL in the core fluid, its viscosity is too high and its fluidity is poor, resulting in frequent needle clogging during spraying.

[0059] Comparative Example 2 microspheres, observed under an optical microscope, showed significant core collapse, small size, and uneven distribution (see attached image). Figure 7This is because the concentration of the outer fluid is too low, and the shell layer is too thin. After the jet is ejected from the nozzle, it cannot form a sufficiently strong "tube wall" to enclose the core fluid. After the solvent evaporates, the thin shell layer is flattened by atmospheric pressure, causing the internal space to shrink (collapse), and the core is stretched and deformed.

[0060] Test Example 2: Drug Loading and In Vitro Release Study of 6-Gin@GlcNAc@CaO2

[0061] The effect of 6-Gin concentration on drug loading of microspheres was investigated. Microspheres were prepared according to the method of Example 1, except that the amount of 6-Gin in step (2) was changed to 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.8 mg and 1.0 mg, respectively, so that the 6-Gin concentration was increased from 0.2% to 1.0%. All other preparation conditions were the same as in Example 1.

[0062] The drug loading of the prepared microspheres was detected by means of the following method: 10 mg of microspheres were accurately weighed, 1 mL of methanol was added, and the microspheres were ultrasonically broken to fully release the encapsulated 6-Gin. The supernatant was then collected by centrifugation. The concentration of 6-Gin in the supernatant was determined by high performance liquid chromatography, and the drug content was calculated by standard curve.

[0063] The results showed that the drug loading of the microspheres continuously increased with increasing 6-Gin concentration; when the concentration reached 0.6%, the drug loading tended to saturate. Further increasing the concentration to 0.8% resulted in a decrease in drug loading (see attached figure). Figure 8 This may be due to excessive drug exceeding the carrying capacity of the outer shell fluid (chitosan matrix), leading to leakage during preparation or washing. When the feed concentration was increased to 1%, the microspheres exhibited uneven morphology and even rupture (see attached image). Figure 9 Taking into account both drug loading efficiency and microsphere morphology, the optimal 6-Gin concentration was determined to be 0.4%-0.6%. In this study, microspheres were prepared with a 6-Gin concentration of 0.5% (i.e., the scheme in Example 1, with a drug loading of 4.2%) for in vitro release studies.

[0064] Accurately weigh 50 mg of the drug-loaded microspheres prepared in Example 1, disperse them in 5 mL of release medium, and then place them into a dialysis bag. Immerse the dialysis bag in 100 mL of release medium at the corresponding pH, and shake in a constant-temperature shaker at 37°C and 100 rpm. Take a 1 mL sample at a predetermined time point, and simultaneously replenish with an equal amount of fresh release medium at the same temperature. Analyze the sample by HPLC and calculate the cumulative release rate of 6-Gin. The release results are shown in the appendix. Figure 10In a release medium at pH 2.0 (0.2 mol / L hydrochloric acid-potassium chloride solution (self-prepared), simulating the gastric fluid environment), 6-Gin exhibits a burst release (approximately 50%) within 0-2 hours, with a cumulative release rate >90% within 24 hours; while in a release medium at pH 7.4 (phosphate-buffered saline (PBS, P1020, Solarbio), simulating the intestinal or body fluid physiological environment), the cumulative release rate of 6-Gin is <30% within 24 hours.

[0065] Test Example 3: Determination of Oxygen Release Kinetics of 6-Gin@GlcNAc@CaO2

[0066] Five mg of 6-Gin@GlcNAc@CaO2 prepared in Example 1 were added to 50 mL of PBS buffer at pH 2.0 and pH 7.4, respectively, and placed in a 37°C constant temperature water bath. A calibrated dissolved oxygen electrode was inserted into the reaction system and sealed. The dissolved oxygen concentration at different time points was continuously recorded under magnetic stirring. The results show (see attached). Figure 11 In an acidic environment with pH 2.0, the outer shell rapidly disintegrates, exposing the inner core CaO2, which reacts with water to release oxygen. On the first day, the dissolved oxygen concentration rises to 15.18 mg / L, accounting for about 70% of its total oxygen release potential, and can maintain continuous release for more than 72 hours. However, under neutral conditions with pH 7.4, due to the protective effect of the shell, water has difficulty contacting the inner core CaO2, and the dissolved oxygen concentration remains at 6-7 mg / L throughout the monitoring period, with negligible oxygen release.

[0067] Test Example 4: Biosafety Evaluation of 6-Gin@GlcNAc@CaO2 Microspheres

[0068] Human gastric epithelial cells GES-1 were used at a density of 4 × 10⁻⁶ cells per well. 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured at 37°C and 5% CO2 for 24 hours. Then, the medium was replaced with fresh medium containing different concentrations of 6-Gin@GlcNAc@CaO2 microspheres (Example 1) and cultured for another 24 hours. 10 μL of CCK-8 solution was added to each well, and the cells were incubated at 37°C for 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the relative cell viability was calculated. The results showed that even with a microsphere concentration as high as 2000 μg / mL, the relative viability of GES-1 cells remained above 90% (see attached image). Figure 12 This indicates that the microspheres have excellent biocompatibility.

[0069] Test Example 5: Effect of 6-Gin@GlcNAc@CaO2 microspheres on the adhesion of H. pylori

[0070] H. pylori was resuspended in DMEM medium to prepare a solution with a concentration of 4 × 10⁻⁶.6 CFU / mL bacterial suspension; 100 μL of bacterial suspension was added to each experimental group and co-incubated with different concentrations of 6-Gin@GlcNAc@CaO2 microspheres (Example 1); a blank control group and an H. pylori control group were set up; after mixing each group, they were cultured for the corresponding time; then urease test solution was added for color development, and the absorbance was measured at 550 nm using an ELISA reader, and the bacterial adhesion rate was calculated, adhesion rate % = (A 实验组 -A 空白 ) / (A H. pylori对照 -A 空白对照 )×100%. The results show (attached) Figure 13 The microspheres effectively inhibited bacterial adhesion in a concentration- and time-dependent manner. After co-incubation at a concentration of 1000 μg / mL for 24 hours, the bacterial adhesion rate was significantly reduced.

[0071] Test Example 6: The mitigating effect of 6-Gin@GlcNAc@CaO2 microspheres on the microaerobic microenvironment of H. pylori biofilm

[0072] Sterile circular glass coverslips were coated overnight at 4°C with 0.1% gelatin solution. *H. pylori* suspension was inoculated onto the coverslips and cultured under microaerophilic conditions for 2 hours to induce biofilm formation. Different concentrations of 6-Gin@GlcNAc@CaO2 microspheres (Example 1) were co-incubated with the formed biofilm for 2 hours. All groups were then incubated with the hypoxia probe pymonidazole hydrochloride for another 1 hour. The samples were fixed, incubated overnight at 4°C with anti-pymonidazole primary antibody, and then incubated for 1 hour at room temperature in the dark with Cy3-labeled secondary antibody. Biofilm samples were observed and images were acquired under a fluorescence microscope. The results are shown in the appendix. Figure 14 These microspheres can effectively disrupt bacterial biofilm structures and significantly alleviate the microaerophilic microenvironment inside biofilms in a concentration-dependent manner.

[0073] Test Example 7: Evaluation of the in vitro antibacterial activity of 6-Gin@GlcNAc@CaO2 microspheres

[0074] H. pylori was resuspended in DMEM medium to prepare a solution of 1×10⁻⁶. 7 CFU / mL bacterial suspension; in 96-well plates, 50 μL of bacterial suspension was mixed with 50 μL of 6-Gin@GlcNAc@CaO2 microspheres (Example 1) at different concentrations (200, 500, 1000 μg / mL); an equal volume of DMEM medium was used as a negative control instead of the microsphere solution; after co-incubation for 24 hours, the absorbance at 590 nm was measured using a microplate reader, and the growth inhibition rate of the microspheres against H. pylori was calculated. The results show (see attached...). Figure 15 After 24 hours of treatment with microspheres, the growth of H. pylori was significantly inhibited, and the inhibition rate exceeded 80% at a concentration of 1000 μg / mL. This inhibitory effect showed a clear concentration dependence.

[0075] Test Example 8: Analysis of the uptake mechanism of 6-Gin@GlcNAc@CaO2 microspheres

[0076] FITC-labeled 6-Gin@GlcNAc@CaO2 microspheres were prepared; the microspheres prepared in Example 1 were co-incubated with H. pylori suspension for different times; after incubation, the bacterial suspension was collected for flow cytometry analysis; the flow cytometry analysis showed (see attached...). Figure 16 The percentage of bacteria adhering to the microspheres increased with incubation time, demonstrating that the uptake behavior was time-dependent. Another sample was fixed with 4% paraformaldehyde and counterstained with DAPI, followed by observation using a confocal laser scanning microscope. (Confocal microscope images attached) Figure 17 The results clearly show that as the co-incubation time was extended from 2 hours to 6 hours, the adhesion of SYTO9-stained H. pylori (green) to the surface of Cy5-labeled microspheres (red) increased significantly and formed a dense bacterial layer, which visually confirmed that the microspheres could be actively taken up and enriched by bacteria.

[0077] Test Example 9: Determination of the minimum inhibitory concentration of 6-Gin@GlcNAc@CaO2 microspheres

[0078] The minimum inhibitory concentrations (MICs) of 6-Gin@GlcNAc@CaO2 microspheres, free 6-Gin, and amoxicillin against *H. pylori* prepared in Example 1 were determined using the microdilution method. Each drug was dissolved in DMSO to prepare a stock solution of 3200 µg / mL. In a 96-well plate, 160 µL of *H. pylori* bacterial culture and 40 µL of drug stock solution were added to the first column. From the second to the twelfth column, 100 µL of bacterial culture was added to each well beforehand. Then, 100 µL of the mixture was transferred from the first column to the second column, mixed thoroughly, and serially diluted twofold up to the twelfth column. Finally, 100 µL of the mixture was discarded from the twelfth column, forming a continuous concentration gradient of the drug. After incubating the 96-well plate in a microaerophilic environment for 72 hours, the concentration corresponding to the lowest drug concentration at which no bacterial growth was observed visually was the MIC. The results are shown in the attached figure. Figure 18 The results showed that the MIC value of 6-Gin@GlcNAc@CaO2 microspheres against H. pylori was significantly lower than that of free 6-Gin drug, indicating that the microsphereization strategy effectively enhanced its inherent antibacterial activity.

[0079] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microsphere, characterized in that, It includes an oxygen-releasing core based on polycaprolactone and calcium peroxide, and a pH-responsive shell made of chitosan modified with N-acetyl-d-glucosamine and loaded with the natural drug 6-Gin; the shell of the microspheres disintegrates and rapidly releases 6-Gin in the acidic environment of the stomach, while the core reacts with water to continuously release oxygen.

2. The acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres according to claim 1, characterized in that, The microspheres are regular spherical in shape, with a diameter of 20-30 μm, a core diameter of 15-20 μm, and a shell thickness of 5-10 μm.

3. The acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres according to claim 1, characterized in that, In a release medium with pH=2.0, the microspheres exhibit a cumulative release rate of 6-Gin greater than 90% within 24 hours, with a continuous oxygen release time exceeding 72 hours; in a release medium with pH=7.4, the cumulative release rate of 6-Gin is less than 30% within 24 hours.

4. A method for preparing acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Core fluid preparation: Calcium peroxide was ultrasonically dispersed in a dichloromethane solution of polycaprolactone, filtered, and a homogeneous core fluid was obtained. (2) Preparation of shell fluid: Chitosan was dissolved in an aqueous acetic acid solution, N-acetyl-d-glucosamine and drug 6-Gin were added, and the mixture was stirred until completely dissolved to obtain shell fluid; (3) Microsphere preparation and curing: Using a coaxial electrostatic spraying device, the core fluid obtained in step (1) and the outer shell fluid obtained in step (2) are simultaneously pumped out through the inner and outer needles of the coaxial nozzle, respectively, and sprayed into the sodium tripolyphosphate solution under a high voltage electrostatic field for cross-linking and curing. After collection, washing and drying, the microspheres are obtained.

5. The preparation method according to claim 4, characterized in that, In step (1), the mass concentration of calcium peroxide in the core fluid is 8%-12%, and the mass concentration of polycaprolactone is 6%-10%.

6. The preparation method according to claim 4, characterized in that, In step (2), the mass fraction of the acetic acid aqueous solution is 0.6-1.2%, the mass concentration of chitosan in the shell fluid is 1.5%-2.5%, the mass concentration of N-acetyl-d-glucosamine is 8%-12%, and the mass concentration of 6-Gin is 0.4%-0.6%.

7. The preparation method according to claim 4, characterized in that, In step (3), the parameters of the coaxial electrostatic spray are: voltage 10-15 kV, core fluid flow rate 0.3-0.5 mL / h, outer fluid flow rate 1.5-2.5 mL / h, receiving distance 12-18 cm, inner needle inner diameter 0.15-0.2 mm, outer diameter 0.3-0.35 mm, outer needle inner diameter 0.6-0.65 mm, outer diameter 1.1-1.15 mm.

8. The preparation method according to claim 4, characterized in that, In step (3), The sodium tripolyphosphate solution has a mass fraction of 0.8%-1.2%; The conditions for cross-linking curing are: room temperature, 5-15 min; The drying conditions were: pre-freezing at -80℃ for 24 h, followed by vacuum freeze-drying for 48 h.

9. The use of acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres as described in any one of claims 1-3, or acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres prepared by any one of claims 4-8, in the preparation of drugs or foods for treating Helicobacter pylori or relieving gastritis.

10. The application according to claim 9, characterized in that, The acid-responsive oxygen-releasing chitosan / polycaprolactone core-shell microspheres can inhibit Helicobacter pylori adhesion, disrupt its biofilm structure, alleviate the hypoxic microenvironment inside the biofilm, and exhibit growth-inhibiting activity against Helicobacter pylori.

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