Nanocapsule-loaded in-situ gel for treating helicobacter pylori infection and reversing gastrointestinal metaplasia and application of nanocapsule-loaded in-situ gel

Abstract

The invention belongs to the technical field of biological medicine, discloses nanocapsule-loaded in-situ gel for treating H. pylori infection and reversing intestinal metaplasia of gastric tissue and application of the nanocapsule-loaded in-situ gel, and particularly discloses nanocapsule-loaded in-situ gel which comprises a nanocapsule and pH-responsive gel, the nanocapsule is dispersed in the gel, and the pH-responsive gel is a pH-responsive gel. Comprising an active drug, a macromolecular substance for coating the active drug and a structure stabilizer. According to the nanocapsule-loaded in-situ gel provided by the invention, the nanocapsule-loaded in-situ gel is directionally released on one side by responding to the change of the pH value of an H.pylori infected part, and the nanocapsule responds to lipase secreted by H.pylori to accurately release an active drug, so that efficient sterilization is realized, and damage caused by infection is relieved. Meanwhile, the gel can also effectively reverse gastrointestinal metaplasia caused by H.pylori infection, further reduces the risk of gastric cancer, and provides a new thought for gastric cancer prevention.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a nanocapsule-loaded in-situ gel for treating Helicobacter pylori infection and reversing gastrointestinal metaplasia and an application thereof. Background Art

[0002] Gastric cancer is a major global public health issue, affecting 4.9% of the world's population, and effective preventive measures are urgently needed. Among them, Helicobacter pylori (H. pylori) infection is considered to be one of the most important risk factors for gastric cancer. Data show that H. pylori has a very high global infection rate (about 43%), of which 1% to 2% of H. pylori infected people will develop gastric cancer. Studies have shown that more than 60% of malignant gastric cancer cases are related to H. pylori infection.

[0003] H. pylori infection can lead to an imbalance in the gastrointestinal microenvironment, triggering acute and chronic inflammation, which in turn leads to host genomic instability and may ultimately develop into gastric cancer. The host inflammatory response is mediated by molecules associated with oxidative stress, including excessive proinflammatory cytokines and reactive oxygen species (ROS). Furthermore, H. pylori can inject virulence factors into host cells, interacting with multiple signaling pathways and directly promoting the malignant transformation of gastric cells.

[0004] Currently, the global clinical treatment regimen for H. pylori infection is based on triple or quadruple therapy with antibiotics and proton pump inhibitors (PPIs) without or with bismuth. The latest data show that the eradication rate of H. pylori with clarithromycin-based triple therapy has dropped from 90% in the 1990s to 70% today. At the same time, a growing number of studies have shown that these antibiotic-based clinical therapies can also severely disrupt the balance of gastrointestinal microbiota, potentially delaying the recovery of a healthy microbial environment and even causing other gastrointestinal problems. Therefore, it is crucial to develop a treatment method that can effectively eliminate H. pylori without inducing drug resistance or affecting the microbiome.

[0005] Although there is evidence that eradication of H. pylori can reduce the risk of gastric cancer, it may be ineffective in inhibiting the development of gastric cancer once the disease has progressed to the intestinal metaplasia (GIM) stage. GIM refers to the transformation of the gastric mucosa into an intestinal-type phenotype, often accompanied by long-term H. pylori infection, and is considered a precursor to gastric cancer. Studies have shown that eradication of H. pylori can effectively prevent the development of gastric cancer before the onset of GIM. However, once the disease progresses to the GIM stage, the preventive effect of H. pylori eradication becomes less significant. This means that once GIM develops, reversal is difficult. Currently, only a few traditional Chinese medicines, such as Weierning's, have been shown to have significant efficacy in reversing GIM, but their active ingredients and specific mechanisms of action are unclear. Furthermore, researchers both domestically and internationally are attempting to reverse GIM by modulating the gastric mucosal microenvironment, intervening in molecular signaling pathways, or utilizing tissue engineering techniques. Through animal experiments, some researchers have found that certain growth factors (such as epidermal growth factor (EGF) and fibroblast growth factor (FGF)) can promote gastric mucosal repair and improve GIM to a certain extent. Vitamin D and its derivatives have been found to regulate the differentiation process of gastric mucosal epithelium, helping to inhibit the development of GIM. In addition, manipulating the gastric mucosal microenvironment, such as regulating the structure of the intestinal flora, improving gastric acid secretion, and targeted regulation of related inflammatory factors (such as IL-1β and TNF-α), are all considered to have potential effects on reversing GIM. Currently, there is no clear clinical method to reverse GIM.

[0006] In summary, while existing clinical therapies have made some progress in treating H. pylori infection and the related diseases it causes, they still have significant shortcomings when it comes to H. pylori infection accompanied by GIM, which presents a potential risk of cancer. Existing clinical treatments are unable to effectively reverse GIM while simultaneously eradicating H. pylori, and they also face challenges such as decreased efficacy due to drug resistance and imbalances in the gastrointestinal microbiome. Therefore, it is particularly urgent to develop a new and highly effective treatment strategy that can both eradicate H. pylori and effectively reverse GIM, while avoiding the problems of intestinal microbial imbalance caused by traditional antibiotic therapies. Summary of the Invention

[0007] The first aspect of the present invention aims to provide a nanocapsule.

[0008] The second aspect of the present invention aims to provide an in-situ gel containing nanocapsules.

[0009] The third aspect of the present invention aims to provide a method for preparing the nanocapsule-loaded in-situ gel according to the second aspect of the present invention.

[0010] The fourth aspect of the present invention aims to provide an application of the nanocapsules of the first aspect of the present invention or the nanocapsule-loaded in-situ gel of the second aspect of the present invention.

[0011] The fifth aspect of the present invention aims to provide a pharmaceutical composition.

[0012] The sixth aspect of the present invention aims to provide a pharmaceutical preparation.

[0013] In order to achieve the above object, the technical solution adopted by the present invention is:

[0014] A first aspect of the present invention provides a nanocapsule, wherein the nanocapsule comprises an active drug, a polymer substance for encapsulating the active drug, and a structural stabilizer.

[0015] In some embodiments of the present invention, the active drug includes at least one of curcumin, eugenol, resveratrol, apigenin, glycyrrhetinic acid, thymol, forsythiaside, fagopyrum serrata, and tea polyphenols.

[0016] In some embodiments of the present invention, the active drug comprises curcumin and eugenol.

[0017] In some embodiments of the present invention, the mass ratio of curcumin to eugenol is 1:(4-50); further 1:(10-20).

[0018] In some embodiments of the present invention, the mass ratio of the active drug to the polymer substance is 1:(3-10), further 1:(3-6).

[0019] In some embodiments of the present invention, the mass ratio of the structural stabilizer to the polymer substance is 1:(2-10), further 1:(4-9).

[0020] In some embodiments of the present invention, the high molecular weight substance is lipase-sensitive.

[0021] In some embodiments of the present invention, the polymer substance includes polycaprolactone-polyethylene glycol.

[0022] In some embodiments of the present invention, the nanocapsules are prepared by a preparation method comprising the following steps: an active drug, a structural stabilizer and a polymer substance are dissolved in an organic solvent, and then added dropwise to the solvent under stirring to obtain nanocapsules.

[0023] In some embodiments of the present invention, the organic solvent includes at least one of acetone, ethanol, methanol, dichloromethane, chloroform, and ether.

[0024] In some embodiments of the present invention, the stirring temperature is 30-50° C., and the stirring speed is 700-850 rpm.

[0025] The second aspect of the present invention provides a nanocapsule-loaded in situ gel, comprising the nanocapsules of the first aspect of the present invention and a pH-responsive in situ gel, wherein the nanocapsules are uniformly dispersed in the pH-responsive in situ gel.

[0026] In some embodiments of the present invention, the volume ratio of the nanocapsules to the pH-responsive in-situ gel is 1:(1-5).

[0027] In some embodiments of the present invention, the material of the pH-responsive gel includes at least one of alginate, polyacrylate, carbomer, chitosan, gelatin, gellan gum, pectin, and methyl methacrylate.

[0028] The pH-responsive in-situ gel is prepared by a preparation method comprising the following steps: mixing alginate, polyacrylate and a solvent to obtain alginate gel.

[0029] In some embodiments of the present invention, the mass ratio of alginate to polyacrylate is 1:1-6.

[0030] In some embodiments of the present invention, the alginate includes at least one of sodium alginate, calcium alginate, potassium alginate, ammonium alginate and magnesium alginate.

[0031] In some embodiments of the present invention, the polyacrylate comprises at least one of sodium polyacrylate, copper polyacrylate, zinc polyacrylate, potassium polyacrylate, ammonium polyacrylate and aluminum polyacrylate.

[0032] In some embodiments of the present invention, the solvent includes at least one of water, PBS buffer, and HEPES buffer.

[0033] The present invention develops a pH-responsive drug delivery system based on curcumin (Cur) and eugenol (Eug) as the main active ingredients. By combining sodium alginate (ALG) and sodium polyacrylate (PAANa) gel with lipase-sensitive polycaprolactone-polyethylene glycol material, a nanocapsule-loaded in-situ gel CurE-gel with gastric microenvironment responsiveness was constructed. CurE-gel can respond to changes in the pH value of gastric acid and the action of lipase secreted by H. pylori, achieving unilateral directional release of drugs on the gastric mucosal side, reducing its exposure and leakage in gastric juice. CurE-gel can regulate the release of drugs in response to the elevated pH value of the gastric mucosal lesion site and the lipase activity secreted by H. pylori. This microenvironment-responsive system not only significantly enhances the retention capacity of nanocapsules in the gastric mucosal lesion site, but also improves the targeting of drugs through unilateral directional release, thereby achieving an increase in local drug concentration and an enhancement of therapeutic effect. Furthermore, CurE-gel can effectively eradicate H. pylori and, through its antioxidant properties, eliminate the excessive ROS caused by H. pylori, while also reversing gastrointestinal metaplasia. This mechanism provides new insights into the prevention of gastric cancer.

[0034] CurE-gel can be used to eradicate H. pylori and reverse gastrointestinal metaplasia (GIM), improving gastrointestinal health and reducing the risk of gastric cancer. This invention avoids the damage to the gastrointestinal microbiome caused by traditional antibiotic treatments, providing a non-antibiotic treatment option for GIM. CurE-gel leverages the antimicrobial effects of natural polyphenols such as curcumin and eugenol to address antibiotic resistance, restore the balance of the gastrointestinal microbiome, and minimize side effects.

[0035] Animal studies have shown that CurE-gel has no significant toxic side effects and exhibits high biocompatibility. This system can maintain stable drug release in the stomach for extended periods without interfering with normal gastrointestinal function, demonstrating its high potential for clinical application.

[0036] The third aspect of the present invention provides a method for preparing the nanocapsule-loaded in situ gel of the first aspect of the present invention, comprising the following steps: mixing nanocapsules and pH-responsive in situ gel to obtain the nanocapsule-loaded in situ gel.

[0037] The fourth aspect of the present invention provides the use of the nanocapsule of the first aspect of the present invention or the nanocapsule-loaded in-situ gel of the second aspect of the present invention in at least one of (1) to (9):

[0038] (1) Anti-Helicobacter pylori;

[0039] (2) preparing anti-Helicobacter pylori products;

[0040] (3) Preparation of products for treating and / or preventing Helicobacter pylori infection, or preventing and / or treating diseases caused by Helicobacter pylori;

[0041] (4) preparing products for treating and / or preventing gastric precancerous lesions;

[0042] (5) Preparation of products for maintaining the ecological balance of intestinal microorganisms;

[0043] (6) Antioxidant;

[0044] (7) Preparation of antioxidant products;

[0045] (8) preparing drug delivery systems;

[0046] (9) Prepare gastric epithelial cell protective agent.

[0047] In some embodiments of the present invention, the anti-Helicobacter pylori described in (1) to (2) includes at least one of reducing the virulence of Helicobacter pylori, reducing the urease activity of Helicobacter pylori, clearing Helicobacter pylori biofilm, inhibiting Helicobacter pylori biofilm formation, inhibiting Helicobacter pylori adhesion and killing Helicobacter pylori.

[0048] In some embodiments of the present invention, the disease described in (3) includes a gastric disease.

[0049] In some embodiments of the present invention, the gastric disease includes at least one of gastritis, gastric ulcer, duodenal ulcer, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer.

[0050] In some embodiments of the present invention, the gastric precancerous lesions described in (4) include at least one of gastrointestinal metaplasia and dysplasia.

[0051] In some embodiments of the present invention, the nanocapsule-loaded in situ gel achieves the purpose of treating gastric precancerous lesions by reducing the expression levels of MUC2 and CDX2 in gastric precancerous lesion cells, increasing the expression level of MUC1 in gastric precancerous lesion cells, improving the antioxidant capacity of gastric precancerous lesion cells, inhibiting the activity of gastric precancerous lesion cells, inhibiting the cloning ability of gastric precancerous lesion cells, repairing DNA damage in gastric precancerous lesion cells, and / or inhibiting the proliferation ability of gastric precancerous lesion cells.

[0052] In some embodiments of the present invention, the antioxidant effects of (6) to (7) include reducing the content of ROS and malondialdehyde, and increasing the activities of superoxide dismutase, catalase, and peroxidase.

[0053] In some embodiments of the present invention, the products include reagents, kits, drug delivery systems, and drugs.

[0054] The fifth aspect of the present invention provides a pharmaceutical composition comprising the nanocapsules of the first aspect of the present invention or the nanocapsule-loaded in-situ gel of the second aspect of the present invention.

[0055] In some embodiments of the present invention, the drug has at least one of the following functions: anti-Helicobacter pylori; treatment and / or prevention of Helicobacter pylori infection or prevention of diseases caused by Helicobacter pylori; treatment and / or prevention of gastric precancerous lesions; maintenance of intestinal microbial ecological balance; anti-oxidation; drug delivery.

[0056] In some embodiments of the present invention, the pharmaceutical composition further comprises a drug for treating and / or preventing Helicobacter pylori infection, or preventing and / or treating a disease caused by Helicobacter pylori.

[0057] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0058] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integrators, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoaming agents, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, and carriers.

[0059] In some embodiments of the present invention, the dosage form of the drug includes a dosage form for gastrointestinal administration.

[0060] In some embodiments of the present invention, the dosage form for gastrointestinal administration includes at least one of powders, tablets, granules, capsules, sustained-release agents, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets.

[0061] In some embodiments of the present invention, the dosage forms for administration through the gastrointestinal tract include but are not limited to enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, dispersible tablets, suckable tablets, chewable tablets, effervescent tablets, scratched tablets, sustained-release and controlled-release dosage forms, sustained-release tablets, sustained-release coated tablets, etc.

[0062] A sixth aspect of the present invention provides a pharmaceutical preparation comprising curcumin and eugenol.

[0063] In some embodiments of the present invention, the mass ratio of curcumin to eugenol is 1:(4-50); further 1:(10-20).

[0064] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0065] The beneficial effects of the present invention are:

[0066] The present invention provides a nanocapsule (CurE-cap) that can effectively remove H. pylori biofilms, effectively penetrate the interior of the biofilm, and destroy the biofilm structure by removing proteins and polysaccharides within the EPS, killing bacteria within the biofilm to achieve an antibacterial effect. Treatment with this CurE-cap can significantly reduce H. pylori urease activity and the expression of urease-related virulence factors, thereby reducing the virulence of H. pylori. Furthermore, this CurE-cap exhibits strong antioxidant capacity, significantly reducing lipid peroxidation and the degree of DNA damage. It can also inhibit the carcinogenesis of gastric epithelial cells and slow the progression of gastric cancer.

[0067] The nanocapsule-loaded in situ gel (CurE-gel) provided by the present invention demonstrates significant advantages in treating Helicobacter pylori (H. pylori) infection and gastrointestinal metaplasia (GIM) through multiple mechanisms. It can unilaterally and directionally release the CurE-cap in response to changes in gastric acid pH. The CurE-cap is then degraded by lipase secreted by H. pylori, precisely releasing the drug and achieving targeted, site-specific delivery. This CurE-gel not only eradicates H. pylori but also effectively reverses GIM caused by H. pylori, further reducing the risk of gastric cancer and providing new insights into gastric cancer prevention.

[0068] Its beneficial effects are reflected in the following aspects:

[0069] (1) Effectively eradicate H.pylori

[0070] CurE-gel encapsulates curcumin and eugenol in lipase-sensitive polycaprolactone-polyethylene glycol (PCGL) nanocapsules and enables precise drug delivery via a pH-responsive alginate in situ gelation system, enabling targeted, site-specific treatment of H. pylori-infected areas. Due to the pH-responsive and bioadhesive properties of the alginate gel, CurE-gel instantly gels upon contact with gastric fluid and rapidly adheres to the gastric mucosal surface, prolonging drug retention in the stomach. The PCGL nanocapsules then precisely release curcumin and eugenol to the infected site in response to lipase secreted by H. pylori. Curcumin and eugenol effectively eradicate H. pylori through multiple mechanisms, including reducing H. pylori virulence, decreasing H. pylori urease activity, clearing H. pylori biofilms, killing dispersed bacteria within biofilms, inhibiting H. pylori adhesion, and directly eliminating H. pylori.

[0071] (2) Reversal of GIM

[0072] CurE-gel can significantly alleviate and reverse GIM caused by long-term chronic infection with H. pylori. In the case of GIM, the gastric mucosal epithelium transforms to an intestinal type. CurE-gel eliminates the excessive reactive oxygen species (ROS) caused by H. pylori infection through its antioxidant activity, thereby alleviating the induced cell damage, including lipid peroxidation and DNA damage, thereby helping to restore the normal structure of the gastric mucosal epithelium. In addition, CurE-gel can regulate key biomarkers related to GIM (MUC1, MUC2 and CDX2) to normal levels, achieve GIM reversal, and reduce the risk of gastric cancer.

[0073] (3) Restore gastric microecological balance

[0074] CurE-gel can not only effectively eliminate H. pylori, but also avoid disrupting the gastrointestinal microecological balance during treatment, which is an unavoidable side effect of traditional antibiotic therapy. Traditional antibiotic therapy affects the abundance of various gastrointestinal flora, leading to gastrointestinal microecological imbalance and increasing the risk of gastric cancer. CurE-gel treats H. pylori infection by delivering non-antibiotics, greatly reducing the impact on the flora. Animal experimental results show that CurE-gel can restore the abundance of beneficial bacteria to a healthy state, and its effect is better than traditional quadruple therapy. By restoring the balance of the gastric microbiome, CurE-gel effectively reduces the risk of gastrointestinal diseases and gastric cancer caused by microbial imbalance.

[0075] (4) Unilateral directional release and targeted delivery

[0076] CurE-gel uses multiple mechanisms to achieve unilateral directional release and targeted delivery of drugs. After oral administration, the pH-responsive in-situ gel in the CurE-gel system instantly gels upon contact with gastric juice and sinks to the surface of the gastric mucosa and rapidly adheres. The gel on the adhesion side responds to the increased pH at the lesion site, and its structure becomes loose, enabling unilateral directional release of the nanocapsule CurE-cap in the gel toward the gastric mucosa, improving the targeting of the drug, increasing local drug concentration, and enhancing the therapeutic effect. In addition, the lipase sensitivity of CurE-cap ensures that curcumin and eugenol can be released under the action of the lipase secreted by H. pylori, precisely acting on the infected area in the gastric mucosa, further improving the targeting and therapeutic effect of the drug. This targeted delivery method not only increases the effective concentration of the drug, but also improves its effectiveness in treating H. pylori infection and GIM.

[0077] (5) High safety and low toxicity and side effects

[0078] CurE-gel has significant safety advantages. In both in vivo and in vitro experiments, CurE-gel showed high biocompatibility and low toxic side effects. Compared with traditional antibiotic treatments, CurE-gel does not cause serious damage to the gastric microbiome, nor does it show obvious toxic reactions. It is constructed from natural biomaterials (such as sodium alginate and sodium polyacrylate) and lipase-sensitive materials (such as polycaprolactone-polyethylene glycol). It is not only biodegradable and highly safe, but also can reduce interference with other gastrointestinal functions through precise delivery. In vivo imaging and organ and blood biochemical analysis results show that CurE-gel has good safety and can remain in the stomach to exert its effect, showing good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 Screening of structural stabilizers in CurE-cap formulations; where a is the particle size measured at different mass ratios of LaA and PCL-PEG, and b is the particle size measured at different mass ratios of EHM and PCL-PEG.

[0080] Figure 2 Evaluation of the morphology and stability of CurE-cap; wherein, a is the particle size of CurE-cap, b is the Zeta potential of CurE-cap, c is the morphology of CurE-cap, d is the stability of CurE-cap at different dilutions, e is the size stability of CurE-cap in H. pylori culture medium BHI and cell culture medium DMEM, f is the size stability of CurE-cap in artificial gastric fluid, and g is the storage stability of CurE-cap at room temperature (25°C) and 4°C.

[0081] Figure 3 Evaluation of the morphology, in vitro adhesion, and release rate of CurE-gel; where a represents the adhesion of RhB@ALG-PAANa gel to the gastric mucosal surface, b represents the pores of CurE-gel at the gastric injury site at different pH levels, and c represents the cumulative release rate of the drug from ALG-PAANa gel.

[0082] Figure 4 CurE-cap mucin aggregation rate (a), apparent permeability coefficient (b), and release rate of Cur after incubation with lipase (c).

[0083] Figure 5is the sensitivity of H. pylori to different drugs; among them, a is the antibacterial effect of different concentrations of Eug on H. pylori, b is the antibacterial effect of different concentrations of Cur on H. pylori, c is the fractional inhibitory concentration index (fractional inhibitory concentration index) of the combined antibacterial effect of Eug and Cur on H. pylori, and d is the antibacterial effect of CurE-cap on H. pylori.

[0084] Figure 6 Figure 4 is the effect of different drugs on the removal of H. pylori biofilm; a to d are the evaluation of the effect of different drugs on the removal of H. pylori biofilm by crystal violet staining, a is the treatment with free Eug, b is the treatment with free Cur, c is the treatment with the physical mixture of Eug and Cur, d is the treatment with CurE-cap, and e is the scanning electron microscopy observation of the removal effect of different drugs on H. pylori biofilm, and the scale is 30 μm.

[0085] Figure 7 Evaluation of the effects of different drugs on biofilm disruption and bacterial clearance. (a) shows the penetration of different drugs into H. pylori biofilms. H. pylori were labeled with DAPI (blue fluorescence) and DiD was loaded into nanocapsules (red fluorescence). Scale bar: 20 μm. (b) shows the clearance of extracellular polymers from biofilms by different drugs. (c) shows the survival rate of bacteria in H. pylori biofilms after treatment with different drugs. Live bacteria were labeled with SYTO9 (green fluorescence) and dead bacteria were labeled with PI (red fluorescence). Scale bar: 20 μm.

[0086] Figure 8 The results show that different drugs inhibit the adhesion of H. pylori to the surface of gastric epithelial cells. The scale bar is 20 μm.

[0087] Figure 9 Inhibition of H. pylori urease activity by different drugs. In the figure, ** represents P < 0.01.

[0088] Figure 10 The expression of virulence factors of H. pylori after drug treatment; a~o are the expression of virulence factors UreA (a), UreB (b), FlaA (c), FliD (d), HpaA (e), BabA (f), SabA (g), AlpA (h), AlpB (i), OipA (j), VacA (k), CagA (l), CagE (m), HtrA (n) and GGT (o) after drug treatment of H. pylori, respectively. In the figure, * represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001.

[0089] Figure 11 The toxicity of the drug to gastric epithelial cells.

[0090] Figure 12 Evaluation of drug inhibition of H. pylori virulence factor translocation in gastric epithelial cells. (a) and (b) are the evaluation of drug inhibition of H. pylori virulence factor translocation in gastric epithelial cells, respectively. In the figure, ** represents P < 0.01, and *** represents P < 0.001.

[0091] Figure 13 Fluorescent dye staining was used to observe the cell morphology of gastric epithelial cells infected with H. pylori and after drug treatment. The scale bar is 20 μm.

[0092] Figure 14 This is an investigation of the ability of drugs to alleviate cellular oxidative stress; among them, a is the ability of different treatment groups to alleviate cellular oxidative stress, b is the data statistical graph of a, c~f are the activity detection results of MDA (c), SOD (d), CAT (e) and GPx (f) in GES-1 cells after different treatment groups. In the figure, * indicates P < 0.05 compared with Blank, ** indicates P < 0.01 compared with Blank, *** indicates P < 0.001 compared with Blank; # indicates P < 0.05 compared with Model, ## indicates P < 0.01 compared with Model, ### indicates P < 0.001 compared with Model.

[0093] Figure 15 To evaluate the drug's ability to slow down cell DNA damage; where a is the electrophoresis diagram of cell DNA, and b is the ratio of DNA head to tail content.

[0094] Figure 16 The drug reverses the intestinal metaplasia of cells; a to c are the relative expression levels of MUC1 (a), MUC2 (b) and CDX2 (c) in GES-1 after treatment with different treatment groups. In the figure, * indicates P < 0.05 compared with Untreated, ** indicates P < 0.01 compared with Untreated, *** indicates P < 0.001 compared with Untreated; # indicates P < 0.05 compared with Model, ## indicates P < 0.01 compared with Model, ### indicates P < 0.001 compared with Model.

[0095] Figure 17 The results show the ability of drugs to inhibit cell carcinogenesis. The scale bar is 400 μm.

[0096] Figure 18 Figure 2 is the modeling flow chart (a) and the weight records of mice during the process (b).

[0097] Figure 19 Figure 2 is the H&E sections of important organs, organ indices, liver blood biochemical indices and kidney function indices; among them, a is the H&E sections of important organs, the scale of heart / spleen / lung is 125 μm, and the scale of liver / kidney is 275 μm, b~f are the organ coefficients of the main organ indices of mice in each treatment group, such as heart (b), liver (c), spleen (d), lung (e) and kidney (f), g~j are the activity detection results of blood biochemical indices of mice in each treatment group, such as ALT (g), AST (h), CRE (i) and BUN (j), respectively. In the figure, * represents P < 0.05.

[0098] Figure 20 The remaining amount of H. pylori in the stomach (a) and the detection of H. pylori in the stomach and pathological conditions by H&E staining (b). In the figure, * indicates P < 0.05 compared with the Model, ** indicates P < 0.01 compared with the Model, *** indicates P < 0.001 compared with the Model; # indicates P < 0.05 compared with Eug+Cur, ## indicates P < 0.01 compared with Eug+Cur, ### indicates P < 0.001 compared with Eug+Cur; && indicates P < 0.01 compared with Cur@Eug@PCL NC; △ indicates P < 0.05 compared with Quadruple therapy.

[0099] Figure 21 AB-PAS sections of mouse stomach; wherein, a is the AB-PAS staining result of mouse stomach, b to e are the expression levels of TNF-α (b), NF-κB (c), COX2 (d) and p53 (e) in the gastric tissue of mice in each treatment group, respectively. In the figure, ** indicates P < 0.01 compared with Healthy; # indicates P < 0.05 compared with Model.

[0100] Figure 22 The abundance distribution (a) and quantitative analysis (b) of gastric microbiota in mice at the phylum level. DETAILED DESCRIPTION

[0101] The present invention is further described in detail below through specific examples.

[0102] It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0103] To make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer are used. Where the manufacturer of the reagents or instruments is not specified, all are conventional products that can be purchased commercially.

[0104] The features and performance of the present invention are further described in detail below with reference to the embodiments.

[0105] Example 1 Preparation and characterization of nanocapsule-loaded in situ gel (CurE-gel)

[0106] This embodiment provides a nanocapsule-loaded in situ gel (CurE-gel). CurE-gel encapsulates curcumin (Cur) and eugenol (Eug) in lipase-sensitive polycaprolactone-polyethylene glycol (PCL-PEG) nanocapsules and achieves precise drug delivery through a pH-responsive sodium alginate (ALG) gel system, thereby achieving targeted treatment in H. pylori-infected areas.

[0107] A method for preparing a nanocapsule-loaded in-situ gel (CurE-gel) comprises the following steps:

[0108] (1) Preparation of CurE-cap

[0109] Curcumin, eugenol, lauric acid (LaA) and polycaprolactone-polyethylene glycol (PCL-PEG, purchased from Jinan Daigang Bioengineering Co., Ltd., product number 2022081706) were dissolved in acetone at a mass ratio of 1-5:20-50:1-10:1-10 (the optimal mass ratio PCL-PEG:LaA:Eug:Cur=9:1:30:1.5) to obtain an acetone mother liquor; under the conditions of stirring speed 800 rpm and stirring temperature 30-50°C, the acetone mother liquor was added dropwise to a deionized water solution, and stirred until the acetone was evaporated to obtain organic solvent-free nanocapsules CurE-cap, wherein the volume ratio of deionized water to acetone was 30:1.

[0110] (2) Preparation of nanocapsule-loaded in situ gel (CurE-gel)

[0111] Sodium alginate (ALG, purchased from Qingdao Mingyue Seaweed Co., Ltd.) was dispersed and dissolved in deionized water and defoamed overnight at 4°C to obtain an ALG stock solution (concentration: 1.6%). To maintain a fixed gel concentration of 1.6%, the ALG solution and sodium polyacrylate (PAANa, purchased from Tianjin Fuchen Chemical Reagent Co., Ltd., TM 604) were placed in a 45°C magnetic water bath at a ratio of 1:1 to 1:6 (optimal mass ratio: 1:3) and stirred for 1-10 minutes to obtain a blank ALG-PAANa gel (ALG-PAANagel). CurE-cap was added (volume ratio of ALG-PAANa gel to CurE-cap was 2:3) to obtain CurE-gel.

[0112] The type and amount of structural stabilizer in the nanocapsule (CurE-cap) were optimized as follows:

[0113] The appearance, particle size, and polymer dispersity index (PDI) during preparation were used as indicators to determine whether a structural stabilizer was added, as well as the optimal ratio of PCL-PEG in the CurE-cap shell to the optimal structural stabilizer. As shown in Table 1, when a structural stabilizer or ethyl hydrogen malonate (EHM) was added, no oil droplets precipitated during the preparation process, and the prepared solution was clear. The ratio of LaA or EHM to PCL-PEG was further screened. When LaA was used as a stabilizer and PCL-PEG:LaA=9:1, the measured particle size and PDI were both the smallest ( Figure 1 ), as the best structural stabilizer and used in the optimal ratio.

[0114] Table 1 Screening of structural stabilizers

[0115]

[0116] The morphology of CurE-cap was characterized and its stability was measured. The specific experimental process is as follows:

[0117] Morphology Observation: A copper mesh was placed upside down on a CurE-cap solution for 5 minutes. After removing excess nanocapsules with filter paper, the mesh was placed upside down on a drop of phosphotungstic acid solution for 2 minutes. The excess dye was removed with filter paper and the mesh was dried at room temperature. The morphology was observed using a transmission electron microscope.

[0118] Stability Assay: CurE-cap was incubated with DMEM (containing 10% FBS), H. pylori culture medium, and artificial gastric fluid (containing sodium chloride, dilute acid, and pepsin) at pH 1.0. Particle size distribution was measured using a Malvern particle size analyzer at specific time points to assess the stability of CurE-cap in both culture medium and gastric acid. Furthermore, CurE-cap was stored at 4°C and room temperature, and particle size changes were measured at different times to assess storage stability.

[0119] The in vitro adhesion, gel porosity, and drug release from the ALG-PAANa gel of CurE-gel were determined. The specific experimental procedures are as follows:

[0120] In vitro adhesion assessment: After establishing a C57BL / 6 mouse model, the stomach was dissected and cut open along the greater curvature. The stomach contents were gently washed away with saline. The stomach was then flattened and immersed in artificial gastric fluid. Equal amounts of free RhB (pink) and RhB-encapsulated gel (RhB@ALG-PAANa gel, prepared similarly to CurE-gel, except that the CurE-cap is replaced with RhB) were added dropwise from above the artificial gastric fluid. The stomach was removed from the artificial gastric fluid, and the unadhered free RhB and gel were washed away with running water. The remaining adhesion was then measured.

[0121] Gel porosity evaluation: CurE-gel was placed in pH 1.5 and pH 4.5 environments to form gels, then quickly frozen at -80°C and freeze-dried. Finally, it was attached to a copper sheet and sprayed with gold. Its surface morphology and pores were observed using a scanning electron microscope.

[0122] Drug release from ALG-PAANa gel was measured by dripping RhB@ALG-PAANa gel into a dialysis bag filled with artificial gastric fluid. The bag was then placed in release media of varying pH values (pH 1.5, pH 2.5, pH 3.5, and pH 4.5). The bag was incubated at 37°C and 100 rpm. The RhB release was measured at regular intervals, and the cumulative release rate was calculated.

[0123] This example uses lipase-sensitive amphiphilic material PCL-PEG as the polymer shell, and carries active drugs Cur and Eug to construct nanocapsules (CurE-cap). Figure 2 As shown in Figures a and b, the particle size of the blank nanocapsule E-cap (104.50±0.78nm) is slightly smaller than that of the drug-loaded CurE-cap (146.73±1.46nm). Both E-cap and CurE-cap have weak negative charges. Figure 2As shown in Figure c, both E-cap and CurE-cap exhibit good dispersion and similar morphology. Figure 2 As shown in Figure d, the particle size of CurE-cap only slightly increased with the increase of dilution ratio, indicating that it can be stable after dilution in body fluids after oral administration. Figure 2 As shown in Figures e to f, the size of CurE-cap was stable in H. pylori culture medium BHI, cell culture medium DMEM, and artificial gastric fluid. Figure 2 As shown in Figure g, CurE-cap also exhibits good storage stability at room temperature and 4°C. Therefore, the stability of CurE-cap can meet the needs of subsequent experiments and practical applications.

[0124] The results of the characterization of CurE-gel were as follows Figure 3 As shown in Figure a, compared with free RhB, RhB@ALG-PAANa gel can sink quickly and adhere to the surface of the gastric mucosa. This indicates that after oral administration, liquid ALG-PAANa can form an in situ gel when it encounters gastric fluid and adhere to the gastric mucosa under the action of gastric peristalsis, rather than floating in the gastric fluid. In addition, Figure 3 As shown in Figure b, the pores of CurE-gel increased and enlarged significantly at the site of gastric injury with a higher pH level. Figure 3 As shown in Figure 3c, this results in faster drug release and a higher cumulative release rate. Therefore, the gel can form a protective barrier in the stomach and also serve as a platform for unilateral release of the drug contained in the gel.

[0125] Example 2 Delivery efficiency of CurE-cap

[0126] This example is used to investigate the delivery efficiency of CurE-cap. The specific experimental process is as follows:

[0127] Determination of mucin aggregation rate: 0.4%, 1.0%, 2.5%, and 5.0% mucin solutions were prepared, and coumarin 6 (C6) was loaded into the nanocapsules instead of curcumin to obtain C6@CurE-cap (i.e., the preparation method was the same as CurE-cap, the only difference was that curcumin was replaced by coumarin 6). The nanocapsules were mixed with the mucin solution (1:10) and incubated for 1 hour. After centrifugation at 5000 rpm for 5 minutes, the supernatant (drug not bound to mucin) was collected and the C6 content (fluorescence value) was determined after demulsification. The mucin aggregation rate was calculated according to the following formula.

[0128]

[0129] Apparent permeability coefficient determination: 40 μg of fresh porcine mucus was evenly spread in a Transwell chamber. 700 μL of PBS was added to the lower receiving chamber. After equilibration at 37°C and 100 rpm, C6@CurE-cap was added dropwise on top of the mucus. After incubation at 37°C and 100 rpm for 4 hours, 100 μL of the solution was sampled from the receiving chamber for demulsification and the fluorescence value was measured. The apparent permeability coefficient (Papp) of the mucus was calculated according to the following formula to evaluate the drug's mucus penetration.

[0130]

[0131] Where dQ / dt represents the ratio of the cumulative amount of fluorescent C6-labeled nanovesicles penetrating from the donor chamber to the receiving chamber and the time, C0 represents the initial concentration of nanovesicles in the donor chamber, and A represents the effective penetration area (cm 2 ).

[0132] Drug release from CurE-cap was measured: CurE-cap was incubated with 20 U / mL and 50 U / mL lipase solutions, respectively. Particle size, particle dispersion index (PDI), and particle number were measured using a Malvern particle size analyzer to evaluate structural changes in the CurE-cap in the presence of lipase. Furthermore, the CurE-cap was placed in a dialysis bag to evaluate drug release at pH 1.5 in release media with or without lipase.

[0133] The results showed that after CurE-cap was released from the gel into the mucus, due to its small particle size and negative charge, e.g. Figure 4 As shown in Figures a to b, it exhibits a low aggregation rate with mucin and a high apparent permeability coefficient. Therefore, CurE-cap can effectively penetrate the gastric mucus layer. Figure 4 As shown in (c), CurE-cap responds to the lipase secreted by H. pylori and releases the loaded drug.

[0134] Example 3 Detection of CurE-cap's anti-H. pylori activity in vitro

[0135] This example is used to investigate the in vitro anti-H. pylori activity of CurE-cap. The specific experimental process is as follows:

[0136] Antimicrobial activity assay: The antimicrobial activity of the drug was assayed by broth dilution method. H. pylori was collected and incubated with the drug at the same time. The final concentration of H. pylori was adjusted to OD 600= 0.1, culture medium is BHI (containing 10% FBS). After 72-96 hours of culture, observe the turbidity of the broth. The minimum inhibitory concentration (MIC) is determined if no growth is observed by naked eye.

[0137] Evaluation of drug penetration and distribution in biofilms: H. pylori were inoculated in glass-bottomed culture dishes and cultured for 72 hours to obtain mature H. pylori biofilms. DiD-loaded nanocapsules were prepared and incubated with H. pylori biofilms, respectively, with free DiD and DiD@E-cap (containing equal amounts of DiD). Drug that had not penetrated the biofilms was washed away with PBS after 1 and 2 hours, respectively. DAPI was then added and incubated in the dark for 20 minutes. Excess dye was washed away with PBS, and images were taken using confocal laser scanning microscopy (CLSM) using excitation channels of 405 nm and 644 nm.

[0138] Investigation of surviving bacteria within the biofilm: H. pylori was inoculated into a culture dish (with a glass bottom) and cultured for 72 hours to obtain a mature H. pylori biofilm. Drugs were added (maintaining the same dosage concentration, Cur was 7.66μg / mL and Eug was 145μg / mL) and incubated with the dish for 24 hours. Free bacteria were washed away with PBS. Bacteria within the biofilm were labeled with SYTO9 and PI in the Live / Dead Backlight Bacterial Viability Kit (SYTO9 can label both live and dead bacteria, while PI can only label dead bacteria) and incubated in the dark for 15 minutes. After washing away the excess dye with PBS, the survival of bacteria within the biofilm was observed by CLSM, with excitation channels at 488nm and 561nm.

[0139] Evaluation of extracellular polymeric substances (EPS): Mature H. pylori biofilms were treated with the drug (maintaining consistent dosing concentrations: Cur 7.66 μg / mL, Eug 145 μg / mL). Free bacteria were then washed away with PBS. Remaining biofilms were removed using a cell scraper and ultrasonically disrupted. The resulting fragments were then centrifuged at high speed (11,000 rpm for 15 minutes). The supernatant was filtered through a 0.22 μm filter. The filtrate was dialyzed, freeze-dried, and concentrated. Polysaccharide content was determined by the phenol-sulfuric acid method, and protein content was determined by a BCA assay.

[0140] Evaluation of biofilm removal: Mature H.pylori biofilms were cultured in microplates, and after 24 hours of drug incubation, crystal violet staining was used for detection. This was an evaluation of the effect of a single dose. After 24 hours of drug incubation, the drug was aspirated, fresh drug was added, and then incubated for another 24 hours before crystal violet staining was performed. This was a test of repeated dosing. SEM observation: The cell slides were treated with acid overnight, washed, sterilized, and dried for later use. Mature H.pylori biofilms were cultured on cell slides, treated with drugs (keeping the drug concentration consistent, Cur was 7.66μg / mL, Eug was 145μg / mL), and then fixed by immersion in 2.5% glutaraldehyde overnight. Subsequently, they were dehydrated with ethanol gradient and tert-butanol, freeze-dried, and sprayed with gold. The biofilm structure and removal effect were observed under a scanning electron microscope.

[0141] Evaluation of bacterial adhesion inhibition: Gastric epithelial GES-1 cells were seeded into glass-bottomed culture dishes and cultured overnight until adherent. Cells were infected with H. pylori (labeled with 2 μg / mL CFDA-SE (green fluorescence) for 25 minutes) at an MOI of 1:10 and incubated with the drug for 4 hours. Non-adherent H. pylori were washed away with PBS and incubated with 10 μg / mL of the nuclear dye Hoechst 33528 (blue fluorescence) at room temperature for 5 minutes. After rinsing away excess dye with PBS, the drug's inhibition of H. pylori adhesion to the biological surface was observed using CLSM, with excitation channels at 405 nm and 488 nm.

[0142] Evaluation of Inhibition of Bacterial Urease Activity: The ability of the drug to inhibit H. pylori urease activity was evaluated using the indigo blue colorimetric assay. H. pylori cells incubated with the drug (Cur at 1.92 μg / mL, Eug at 36.25 μg / mL) were collected by centrifugation. After addition of the extract, the cells were sonicated in an ice bath (300 W, 2 seconds with 3 seconds intervals, for a total of 5 minutes) to lyse the H. pylori. The supernatant was then centrifuged at 13,400 rpm for 15 minutes, and assayed using the Urease Activity Assay Kit (Beijing Solebold Technology Co., Ltd.).

[0143] Determination of the regulation of bacterial virulence factor expression: The total RNA of H. pylori after co-incubation with drugs was extracted using the Trizol method, and cDNA was obtained by reverse transcription after removing gDNA. The expression of virulence factors (UreA, UreB, FlaA, FliD, HpaA, BabA, SabA, AlpA, AlpB, OipA, VacA, CagA, CagE, HtrA and GGT) of H. pylori after treatment with different drugs was detected by qPCR. In order to eliminate the difference in bacterial quantity, the Cp value of the target gene was first subtracted from the Cp of 16S rRNA, and then 2-△△Cp The relative expression levels of H. pylori virulence factors were calculated using the PCR method.

[0144] The results are as follows Figures 5 to 10 As shown, Figure 5 As shown in Figure 3, Cur exhibited a stronger antibacterial effect, while the combination of Cur and Eug exhibited a synergistic antibacterial effect. Figure 6 As shown in Figure 2, crystal violet staining and scanning electron microscopy results showed that the biomass of H. pylori biofilms was significantly reduced after drug treatment, and CurE-cap treatment could eliminate most of the H. pylori biofilms. Figure 7 As shown in the figure, CurE-cap can penetrate into the biofilm well and destroy the biofilm structure by removing proteins and polysaccharides in EPS, while killing bacteria inside the biofilm to achieve antibacterial effect. Figure 8 As shown in Figure 2, CurE-cap treatment can effectively inhibit the adhesion of H. pylori to the surface of gastric epithelial cells. Figure 9 and Figure 10 As shown in Figures a to b, CurE-cap treatment can significantly reduce H. pylori urease activity and the expression of urease-related virulence factors. In addition, H. pylori motility ( Figure 10 c~d)、Adhesion( Figure 10 e~j) and pathogenicity ( Figure 10 The expression levels of virulence factors related to k-o were also reduced. Therefore, CurE-cap effectively reduced the virulence of H. pylori.

[0145] Example 4 Evaluation of the protective effect of CurE-cap on gastric epithelial cells

[0146] This example is used to investigate the protective effect of CurE-cap on gastric epithelial cells. The specific experimental process is as follows:

[0147] Evaluation of drug safety on gastric epithelial cells (GES-1): GES-1 cells were seeded into microtiter plates and cultured overnight at 37°C and 5% CO2 until the cells adhered to the wall. Figure 8 ) After culturing for 24 h, the activity of GES-1 was detected by MTT assay.

[0148] Evaluation of inhibition of virulence factor translocation in gastric epithelial cells: GES-1 was cultured in a microplate until adherent, and H. pylori (MOI 20:1) and drugs (Cur was 1.92 μg / mL, Eug was 36.25 μg / mL) were added and incubated for 24 hours. The cell surface was washed multiple times with PBS to remove H. pylori adhered to the cell surface to the greatest extent possible. Total RNA from washed cells was extracted using the Trizol method, and the content of H. pylori virulence factors (VacA, CagA) in GES-1 was detected using H. pylori-specific qPCR primers, with cellular GAPDH as the internal reference. This was used to investigate the ability of drugs to inhibit the translocation of H. pylori virulence factors VacA and CagA in gastric epithelial cells.

[0149] Evaluation of the effect of H. pylori on gastric epithelial cell damage: GES-1 cells were infected with H. pylori at an MOI of 1:20 and incubated with the drugs (maintaining consistent dosing concentrations: Cur at 1.92 μg / mL and Eug at 36.25 μg / mL) for 24 hours. After washing with PBS, the cells were fixed with 4% paraformaldehyde for 30 minutes at room temperature. After washing with PBS, the cells were permeabilized with 0.5% Triton X-100 for 5 minutes. After washing with PBS, the cells were incubated with the cytoskeletal dye phalloidin (red fluorescence, 100 nM) prepared in 1% BSA in PBS for 30 minutes at room temperature. Excess dye was then washed away with PBS. The cells were then incubated with the nuclear dye Hoechst 33528 (blue fluorescence, 10 μg / mL) for 5 minutes at room temperature. After washing away excess dye with PBS, CLSM observations were performed using excitation channels of 405 nm and 561 nm.

[0150] The results show that Figure 11 As shown in Figure 3, neither the free drug nor CurE-cap showed significant cytotoxicity, indicating low in vitro cytotoxicity and good compatibility. Figure 12 As shown in Figure 3, CurE-cap significantly reduced the translocation of CagA and VacA into gastric epithelial cells, mainly through the action of Cur, and effectively reduced the virulence of H. pylori. Figure 13 As shown, H. pylori infection elongates the cytoskeleton, resulting in a spherical cell shape with unclear membrane boundaries. Treatment with CurE-cap successfully alleviated these changes, restoring normal cell morphology. Therefore, CurE-cap effectively protects gastric epithelial cells.

[0151] Example 5 Evaluation of the effect of CurE-cap in alleviating oxidative stress

[0152] This example is used to investigate the effect of CurE-cap in alleviating oxidative stress. The specific experimental process is as follows:

[0153] The ability of drugs to alleviate cellular oxidative stress was assessed by flow cytometry. H. pylori (MOI = 10:1) was co-incubated with GES-1 for 6 hours. Subsequently, the model group continued to be co-incubated with H. pylori, while the treatment group added drugs (Cur at 1.92 μg / mL, Eug at 36.25 μg / mL) and co-incubated with H. pylori for 18 hours. Finally, the cells were incubated with the ROS probe DCFH-DA and analyzed by flow cytometry.

[0154] Assay for cellular lipid oxidative damage: Cell culture, H. pylori infection, and drug treatment (Cur at 1.92 μg / mL and Eug at 36.25 μg / mL) were performed as described above for ROS levels. After PBS washing, malondialdehyde (MDA) content and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities were measured according to the kit instructions (Anhui White Shark Biotechnology Co., Ltd.).

[0155] The results showed that H. pylori infection often leads to excessive production of harmful ROS, which can cause lipid peroxidation and DNA damage. Figure 14 As shown in Figures a-b, CurE-cap treatment can almost restore the generated ROS to normal levels. Figure 14 As shown in Figures d to f, CurE-cap can effectively restore the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (GPx) in cells, and the level of malondialdehyde (MDA) is significantly reduced to normal levels (1.49 μM, Figure 14 Finally, if Figure 15 As shown in the figure, DNA damage was assessed. After H. pylori infection, tail DNA accounted for 22.5% of the total DNA, whereas after CurE-cap treatment, tail DNA accounted for only 2.7%. Therefore, CurE-cap exhibits potent antioxidant capacity, significantly mitigating lipid peroxidation and reducing the extent of DNA damage.

[0156] Example 6 Evaluation of the Effect of CurE-cap on Reversing Intestinal Metaplasia and Preventing Gastric Cancer

[0157] This example is used to investigate the effect of CurE-cap in reversing intestinal metaplasia and preventing gastric cancer. The specific experimental process is as follows:

[0158] Drug reversal test of intestinal metaplasia: GES-1 was injected into the 2×10 5Cells were inoculated into 6-well plates and cultured overnight until adhered. The culture medium was replaced with serum-free DMEM containing 1.95 μg / mL MNNG. After culturing for 6 hours, serum-free DMEM containing drugs (Cur is 1.92 μg / mL, Eug is 36.25 μg / mL) and MNNG (1.95 μg / mL) was used to culture for 18 hours. Total RNA of each group of cells was extracted using a cell RNA rapid extraction kit and reverse transcribed into cDNA. Template cDNA was amplified by QPCR using SYBR Green fluorescent dye. Human GADPH was used as the internal reference gene and 2 -ΔΔCt The relative expression levels of MUC-1, MUC-2 and CDX-2 in each group were calculated to evaluate the reversal effect of drugs on intestinal metaplasia.

[0159] Investigation of the ability to inhibit cell carcinogenesis: A cell clone formation experiment was used to detect the in vitro cloning ability of cells. The blank group consisted of untreated normal gastric epithelial cells, and the modeling group consisted of MNNG-treated gastric epithelial cells. The cells were seeded at 500 cells / well in a 6-well plate, and the medium was changed every 3 days. The operation was gentle to avoid blowing up the cells. When the adherent cells in the blank group generally reached 20 cell clones, the drug was added (keeping the drug concentration consistent, Cur was 1.92μg / mL, Eug was 36.25μg / mL) for incubation, and the fresh medium containing the drug was replaced every other day for 2 weeks. It was fixed with 4% paraformaldehyde and stained with 0.1% crystal violet and observed under a cell imager.

[0160] Detection of the degree of cell DNA damage: Single cell gel electrophoresis was used to detect the degree of DNA damage. GES-1 culture, H. pylori infection, and drug treatment were the same as the above-mentioned ROS level determination method (Example 5). Normal melting point agarose solution (1.0%) and low melting point agarose solution (0.7%) were prepared respectively. The normal melting point agarose solution and the low melting point agarose solution were sequentially mixed with the cell suspension (1×10 4 A mixture of 100 GES-1 cells / mL and a low-melting-point agarose solution was plated onto a glass slide. The prepared slide was immersed in pre-chilled lysis buffer and incubated at 4°C to lyse the cells. The lysed slide was transferred to an electrophoresis solution for DNA expansion, followed by horizontal electrophoresis in an ice bath. The slide was then immersed in a neutralization buffer, stained with PI, and observed under CLSM. DNA content ratios were analyzed using CASP software.

[0161] The results showed that during the process of gastric mucosal cells transforming into precancerous lesions such as GIM, the expression level of mucin 1 (MUC1) decreased, while the expression levels of mucin 2 (MUC2) and caudal type homeobox 2 (CDX2) increased. Figure 16As shown in the results, Cur, Eug, and CurE-cap can all restore the levels of MUC1, MUC2, and CDX2 in GIM model cells. Among them, CurE-cap has the strongest effect, while Eug is more effective than Cur. In addition, the colony formation rate reflects the proliferation ability of cells and can therefore partially reflect the carcinogenicity level of cells. Figure 17 As shown, CurE-cap exhibited the strongest effect in reducing the increase in colony formation in model cells, with the smallest colony size and loose cell connections. Therefore, CurE-cap can inhibit the carcinogenesis of gastric epithelial cells and slow the progression of gastric cancer.

[0162] Example 7 In vivo safety evaluation of CurE-cap and CurE-gel

[0163] This example is used to investigate the in vivo safety of CurE-gel and its nanocapsules CurE-cap. The specific experimental process is as follows:

[0164] Weight monitoring: When constructing a mouse model of gastric cancer precancerous lesions (construction flow chart as shown in Figure 18 During the entire process of a), body weight was measured and recorded every 7 days.

[0165] The specific modeling process is as follows: using H. pylori bacterial solution (10 8 CFU / mL) were gavage-infected for a total of 9 times every other day (modeling group), while the blank healthy group was not gavaged with bacterial solution; from the middle stage of infection, mice were allowed to drink sterile water (blank group) or sterile water containing 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) freely (modeling group); and were allowed to eat SPF-grade mouse feed (blank group) or SPF-grade mouse feed containing ranitidine (modeling group) freely.

[0166] Safety Assessment of Vital Organs: After dosing, mice in each group were weighed and dissected, and their hearts, livers, spleens, lungs, and kidneys were removed. Wet weights were measured, and organ coefficients (the ratio of organ weight to body weight) were calculated. These organs were then fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned using H&E staining. Their structures were then observed using a cell imaging device.

[0167] Blood biochemical markers: Before autopsy, blood was collected from the orbital cavity of each mouse using an anticoagulant tube containing sodium heparin. Plasma was collected after centrifugation and stored at −20°C or assayed for the following: glutamic pyruvic transaminase (GPT or ALT), glutamic oxaloacetic transaminase (GOT or AST), creatinine (CRE), and blood urea nitrogen (BUN). The assay methods are described in the instructions for the corresponding assay kits (all purchased from Nanjing Jiancheng Bioengineering Institute).

[0168] like Figure 18 As shown in Figure b, the weight changes of mice were recorded during the establishment of the mouse model, and the weight of the mice grew slowly after treatment. Figure 19 As shown in (a), H&E staining and pathological analysis of important organs showed no obvious lesions. The main organ indicators of CurE-gel-treated mice were not significantly different from those of healthy mice ( Figure 19 After treatment, the blood biochemical indicators remained within the normal range ( Figure 19 g-j) indicate normal liver and kidney function. Therefore, CurE-gel is safe in mice.

[0169] Example 8 In vivo pharmacodynamic evaluation of CurE-cap and CurE-gel

[0170] This example is used to investigate the in vivo pharmacodynamics of CurE-cap and CurE-gel. The specific experimental process is as follows:

[0171] Evaluation of gastric bacterial clearance effect and intestinal metaplasia level: Model mice (Example 7) were randomly divided into 5 groups: model group, Eug+Cur physical mixture group, CurE-cap group, bismuth-containing quadruple therapy group (CLR+AMX+lansoprazole+potassium citrate) and CurE-gel group. The dosages were: Eug 10.41 mg / kg; Cur 0.55 mg / kg; CLR 0.55 mg / kg; AMX 1.1 mg / kg; lansoprazole 0.033 mg / kg; potassium citrate 0.24 mg / kg. The model group was given the same volume of PBS for 7 consecutive days by gavage. 5 days after the end of the administration, the mouse stomach tissue was dissected and cut along the greater curvature of the stomach. After extracting the gastric tissue genome, specific H. pylori 16S primers were used to detect the remaining amount of H. pylori in the stomach by QPCR; paraffin-embedded sections were used to detect gastric pathological conditions and observe H. pylori; and AB-PAS staining was used to detect the characteristic mucin of gastric intestinal metaplasia.

[0172] After 7 consecutive days of oral administration, the residual load of H. pylori in the stomach of mice was confirmed by qPCR, and the distribution of H. pylori in the stomach was observed by H&E staining. Figure 20 A and Figure 20 As shown in b. Using bismuth-containing quadruple therapy as a positive control, all drugs can significantly reduce the H. pylori load in the stomach of mice. 2.58 CFU) to eliminate H.pylori and bismuth quadruple therapy (remaining 10 2.05 CFU) were comparable, with no significant difference between the two. However, after CurE-cap was loaded into the in situ gel to construct CurE-gel, CurE-gel (remaining 10 0.32 The effectiveness of CurE-gel in clearing H. pylori (CFU) was significantly superior to that of CurE-cap and bismuth quadruple therapy. Furthermore, H&E staining revealed no H. pylori residue in the stomachs of mice after CurE-gel administration, whereas a small amount of residual H. pylori was observed in the bismuth quadruple therapy and CurE-cap groups (indicated by yellow arrows). This demonstrates that CurE-gel has excellent H. pylori clearance in vivo and is superior to CurE-cap and bismuth quadruple therapy.

[0173] like Figure 21 As shown, AB-PAS staining of paraffin sections of gastric tissue revealed no blue-labeled acidic mucus in the gastric mucosa of healthy mice without modeling, while some of the gastric mucosa of modeling mice appeared blue. After a week of administration, a bismuth-containing quadruple therapy failed to improve gastric intestinal metaplasia in modeling mice. However, after administration of CurE-gel, only neutral mucus labeled purple-red was observed in the stomachs of these mice. This suggests that CurE-gel has a good ability to alleviate and reverse gastric intestinal metaplasia in mice, thereby reducing the risk of further development of gastric cancer.

[0174] The distribution of gastric flora was further analyzed at the phylum level. Figure 22As shown. Compared with healthy mice without modeling, the abundance of Firmicutes in the modeling group increased while the abundance of Proteobacteria decreased, which is consistent with the results of the existing technology (LI XX, WONG GL, TO KF, et al. Bacterial microbiota profiling in gastritis without Helicobacter pylori infection or non-steroidal anti-inflammatory drug use [J]. PLoS One, 2009, 4 (11): e7985.), which detected this difference in H. pylori-negative volunteers and H. pylori-positive gastritis patients. After the administration of CurE-gel, the abundance of Firmicutes in the stomach of mice decreased and the abundance of Proteobacteria increased, both of which were relatively similar to the levels in the stomach of healthy mice; and the degree to which the abundance of Proteobacteria was restored by bismuth quadruple therapy was not as good as that of CurE-gel ( Figure 22 ).

[0175] While the embodiments of the present invention have been described in detail above with reference to the accompanying drawings, the present invention is not limited to the embodiments described above. Various modifications may be made within the scope of knowledge possessed by a person skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof may be combined with one another unless there is a conflict.

Claims

1. A nanocapsule comprising an active drug, a polymer substance for coating the active drug, and a structural stabilizer, wherein the active drug comprises at least one of curcumin, eugenol, resveratrol, apigenin, glycyrrhetinic acid, thymol, forsythiaside, fagopyrum serrata, and tea polyphenols.

2. The nanocapsule according to claim 1, characterized in that The active drugs include curcumin and eugenol; Preferably, the high molecular weight substance is lipase sensitive; Preferably, the polymer substance comprises polycaprolactone-polyethylene glycol; Preferably, the structural stabilizer includes at least one of lauric acid, monoethyl malonate, poloxamer, polyvinyl pyrrolidone, lecithin, TPGS, and Tween.

3. The nanocapsule according to claim 2, characterized in that The mass ratio of the active drug to the polymer substance is 1:(3-10); and / or the mass ratio of the structural stabilizer to the polymer substance is 1:(2-10).

4. The nanocapsule according to claim 2, characterized in that The nanocapsules are prepared by a preparation method comprising the following steps: active drugs, structural stabilizers and high molecular weight substances are dissolved in an organic solvent, and then added dropwise to the solvent under stirring conditions to obtain the nanocapsules.

5. A nanocapsule-loaded in-situ gel, comprising the nanocapsules according to any one of claims 1 to 4 and a pH-responsive in-situ gel; Preferably, the material of the pH-responsive in-situ gel comprises at least one of alginate, polyacrylate, carbomer, chitosan, gelatin, gellan gum, pectin, and methyl methacrylate; Preferably, the pH-responsive in-situ gel is prepared by a preparation method comprising the following steps: mixing alginate, polyacrylate and a solvent to obtain alginate gel.

6. The method for preparing the nanocapsule-loaded in-situ gel according to claim 5, comprising the following steps: The nanocapsules are mixed with the pH-responsive in-situ gel so that the nanocapsules are uniformly dispersed in the gel, thereby obtaining the nanocapsule-loaded in-situ gel.

7. Use of the nanocapsule according to any one of claims 1 to 4 or the nanocapsule-loaded in-situ gel according to claim 5 in at least one of (1) to (9): (1) Anti-Helicobacter pylori; (2) preparing anti-Helicobacter pylori products; (3) Preparation of products for treating and / or preventing Helicobacter pylori infection, or preventing and / or treating diseases caused by Helicobacter pylori; (4) preparing products for treating and / or preventing gastric precancerous lesions; (5) Preparation of products for maintaining the ecological balance of intestinal microorganisms; (6) Antioxidant; (7) Preparation of antioxidant products; (8) preparing drug delivery systems; (9) Prepare gastric epithelial cell protective agent.

8. The use according to claim 7, characterized in that The anti-Helicobacter pylori effects described in (1) to (2) include at least one of reducing the virulence of Helicobacter pylori, reducing the urease activity of Helicobacter pylori, clearing Helicobacter pylori biofilm, inhibiting Helicobacter pylori biofilm formation, inhibiting Helicobacter pylori adhesion and killing Helicobacter pylori; and / or, the diseases described in (3) include gastric diseases; and / or, the gastric precancerous lesions described in (4) include at least one of gastrointestinal metaplasia and dysplasia; preferably, the gastric diseases include at least one of gastritis, gastric ulcer, duodenal ulcer, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer.

9. A pharmaceutical composition comprising the nanocapsule according to any one of claims 1 to 4 or the nanocapsule-loaded in situ gel according to claim 5; Preferably, the pharmaceutical composition further comprises drugs for treating and / or preventing Helicobacter pylori infection, and preventing and / or treating diseases caused by Helicobacter pylori.

10. A pharmaceutical preparation comprising curcumin and eugenol; Preferably, the mass ratio of curcumin to eugenol is 1:(4-50).

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