A sequential multistage targeted delivery system for repairing intestinal barrier and a preparation method thereof

Abstract

The present application belongs to the technical field of drug delivery, and relates to a sequential multi-stage targeted delivery system of a probiotic and prebiotic combination for repairing intestinal barriers. The delivery system is a microcapsule MP wrapped with NPs; the NPs are solid lipid nanoparticles formed by wrapping a probiotic with hyaluronic acid coupled small molecule acids as a film material; and the shell material of the microcapsule MP is a prebiotic. By wrapping the probiotic with hyaluronic acid coupled small molecule acids as a film material to form NPs, and then wrapping the NPs with a prebiotic wall material to form a microcapsule, the sequential multi-stage targeting and zonal release of different drugs can be achieved; the repair of the barrier will not have a reverse effect; the intestinal epithelial cell proliferation is promoted to repair the mechanical barrier; the intestinal flora abundance is improved to repair the microbial barrier; the mucus secretion is promoted to repair the chemical barrier; and the immune homeostasis is regulated to repair the immune barrier.

Description

Technical Field

[0001] This invention relates to a sequential multi-level targeted delivery system combining postbiotics and prebiotics for repairing the intestinal barrier, belonging to the field of drug delivery technology, and is used to treat various acute and chronic intestinal diseases, including ulcerative colitis, Crohn's disease, irritable bowel syndrome, and chemotherapy-related enteritis. Background Technology

[0002] The adult gut has a surface area of ​​400 square meters, providing a vast interface for interaction between the human body and the external environment. More than 80% of nutrient absorption and waste metabolism occur in the intestinal system, but this frequent exchange of substances significantly increases the risk of invasion by various pathogens and endotoxins. Although the gut has evolved multiple protective barriers (physical, chemical, microbial, and immune barriers) to prevent the entry of harmful substances, factors such as excessive immune stress, antibiotic abuse, chemotherapy side effects, and pathogenic bacterial infections can disrupt the dynamic balance between intestinal microorganisms, epithelial cells, and intestinal immune cells, leading to intestinal barrier damage. Acute intestinal barrier damage allows harmful substances to enter the bloodstream and circulate throughout the body, causing myocarditis, sepsis, and bacteremia. Chronic intestinal barrier damage, due to repeated damage and healing of intestinal epithelial cells, leads to abnormal proliferation of intestinal epithelial cells, significantly increasing the incidence of colorectal cancer. Currently, drugs for treating intestinal diseases fall into two main categories: antibiotics and probiotic preparations. Antibiotics kill pathogenic bacteria but also cause the death of a large number of beneficial bacteria, worsening the microbial barrier. Furthermore, the lysis and death of large numbers of bacteria release endotoxins, inducing a cytokine storm and accelerating epithelial cell apoptosis. Although probiotic preparations can regulate gut microbiota, restore immune homeostasis, and promote intestinal epithelial cell proliferation, firstly, live bacteria preparations are susceptible to damage from temperature, oxygen, gastric acid, and bile salts; secondly, in the inflammatory microenvironment of the intestine, many unfavorable factors such as pathogen invasion and maintaining a numerical advantage, inflammatory cell aggregation, release of inflammatory factors, and elevated redox potential make it difficult for orally administered probiotics to colonize, resulting in significant differences in efficacy; finally, probiotics can also cause bacteremia by escaping into the bloodstream through damaged mucosal layers. Therefore, probiotics cannot be used as a primary direct treatment drug.

[0003] With the interdisciplinary development of multi-omics (microbiome and metabolomics), it has been recognized that probiotics regulate and participate in the repair of the intestinal barrier through metabolites. Among these, changes in the levels of three types of probiotic metabolites (metoelasts: short-chain fatty acids (SCFAs), bile salts (BAs), and tryptophan metabolites (IPAs)) play a crucial role in the development of various intestinal diseases. Studies have shown that metabolites first act as antibiotics, inhibiting pathogenic bacteria while causing far less harm to beneficial bacteria; secondly, as immune signaling molecules, they can reduce the expression of macrophage inflammatory factors; and finally, they promote aerobic metabolism by intestinal epithelial cells, consuming oxygen in the intestinal lumen, providing the necessary conditions for the survival of anaerobic probiotics and indirectly promoting their proliferation. However, metabolites also present challenges such as unpleasant odor, poor stability, and the need for high accumulation concentrations for therapeutic effects. Therefore, an increasing number of researchers are investing in the development and invention of drug delivery systems to improve the therapeutic effects of probiotics, antibiotics, and metabolites.

[0004] Research on drug delivery systems for treating enteritis has largely focused on improving the survival rate of probiotics or targeting pathogens to release antibiotics. Jiezhou Pan's research has developed a "nano-armor" coating composed of tannic acid and iron ions, which can protect bacteria from antibiotics. This "nano-armor" protects probiotics from antibiotic damage. The "armored" probiotics have shown the ability to colonize the gastrointestinal tract of rats treated with levofloxacin, significantly reducing antibiotic-associated diarrhea (AAD) induced by levofloxacin and improving some pre-inflammatory symptoms caused by AAD. (A single-cell nanocoating of probiotics for enhanced amelioration of antibiotic-associated diarrhea, DOI: 10.1038 / s41467-022-29672-z); Haibo Mu et al. used xylo-oligosaccharides to couple antibiotics—ciprofloxacin—via disulfide bonds to achieve targeted release of antibiotics into Salmonella, killing pathogens while reducing further damage to the intestinal microbial barrier by antibiotics (Pathogen-targeting glycovesicles as a therapy for salmonellosis, DOI: 10.1038 / s41467-019-12066-z). A small number of people have taken a different approach, abandoning antibiotics and probiotics, using postbiotics as the main treatment drugs, and enhancing their therapeutic effect through drug delivery systems. For example, Zhao Yuanjin et al. dripped a mixture of post-biotics, prebiotics, and calcium chloride into sodium alginate, followed by coagulation and washing, and then microfluidically electrosprayed it into a chitosan solution. The resulting composite microcapsules significantly increased targeted release into the lower gastrointestinal tract, improved the therapeutic effect of post-biotics, and indirectly promoted the proliferation of beneficial bacteria (CN 114099459 A). However, lesions in lower gastrointestinal diseases (such as ulcerative colitis, Crohn's disease, and necrotizing enterocolitis) are often scattered in island-like locations, and the dominant bacterial species in different microenvironments are not entirely the same. Therefore, simple targeting of lower gastrointestinal organs cannot concentrate and effectively release drugs at the intestinal lesions, nor can it fully utilize the advantages of the intestinal flora. Sequential multi-level targeting is a novel technology that can gradually lock onto and narrow the drug release range, and it has been mentioned for the first time in the field of tumor treatment. For example, the tumor sequential multi-level targeting system designed by Yunai Du et al. has the characteristics of targeting tumor tissue—targeting cancer cells in tumor tissue—targeting mitochondria in cancer cells (Sequential Enzyme Activation of a “Pro-Staramine”-Based Nanomedicine to Target Tumor Mitochondria, DOI: 10.1002 / adfm.201904697).However, there are currently no reports of sequential multi-stage targeted drug delivery systems that use loaded postbiotics as treatments for enteritis. Summary of the Invention

[0005] This invention aims to overcome the shortcomings of poor stability of postbiotics and the need for high accumulation concentrations for treatment, and to improve therapeutic efficacy. It designs an oral drug delivery system consisting of two parts: an outer layer and an inner layer. The outer layer is composed of enteric-coated microcapsules made of various prebiotic membrane materials. Upon entering the intestinal lumen, these capsules immediately cleave to release prebiotics and solid lipid nanoparticles from the inner layer. The prebiotics promote the proliferation of beneficial bacteria in the healthy intestinal microenvironment and continuously generate postbiotics to maintain intestinal homeostasis and prevent inflammation. The inner layer uses hyaluronic acid with CD44 targeting as the membrane matrix, and uses a dual-sensitivity inflammatory response to couple small-molecule acid drugs to directly reach the damaged and inflamed sites of the intestine, releasing high concentrations of postbiotics to repair the damaged intestinal barrier. Through this treatment strategy of different therapies for different sites and a comprehensive approach, the system ultimately achieves self-sufficiency of postbiotics in the intestine, eradicating invading pathogens at the lesion site while reducing harm to innocent bacteria and repairing damaged intestines.

[0006] To achieve the above objectives, the present invention adopts the following specific technical solution:

[0007] This invention provides a sequential multi-level targeted drug delivery system for repairing the intestinal barrier. The delivery system is microcapsules (MPs) encapsulating prebiotics (NPs). The NPs are solid lipid nanoparticles formed by encapsulating prebiotics with hyaluronic acid coupled with small molecule acids as the membrane material. The shell material of the microcapsules (MPs) is a prebiotic.

[0008] In the above technical solution, the small molecule acid is further selected from one or more of acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, isopropionic acid, isobutyric acid, isovaleric acid, lactic acid, citric acid, malic acid, and fumaric acid; the postbiotic is tryptophan metabolite IPAs and bile salt metabolite Bas, wherein IPAs are one or more of indole-3-methanol, indole-3-carboxaldehyde, indolepropionic acid, and diindolemethane, and Bas is one or more of ursodeoxycholic acid, glycocholic acid, glycochenodeoxycholic acid, taurocholic acid, and taurochenodeoxycholic acid; and the prebiotic is one or more of fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, isomaltooligosaccharides, soybean oligosaccharides, sodium alginate, inulin, and chitosan.

[0009] In the above technical solution, the preparation method of the membrane material further includes the following steps:

[0010] (1) Dehydration cyclization synthesis of glycidyl oxide xEpo: Small molecule acid salts are added to freshly distilled epichlorohydrin, an alkaline catalyst is added, and the mixture is heated to reflux. After reacting for 2-24 hours, the reaction is stopped, excess epichlorohydrin is removed from the reaction product, and xEpo is obtained by column chromatography. The preferred mobile phase is V ethyl acetate: V petroleum ether = 1:2-1:20.

[0011] (2) Synthesis of thioacetal / ketone Tx by carbonyl addition reaction: The mercapto compound and the natural aldehyde / ketone compound are reacted at a molar ratio of 2:1-2:2.5 under an acidic atmosphere for 12-48 h. The residual reactants are washed away with saturated brine and organic solvent in sequence, and the product is purified by column chromatography. The natural aldehyde / ketone compound is an aldehyde or ketone compound that is naturally occurring in animals and plants, preferably acetone, butanone, lauraldehyde, citral, benzaldehyde, cinnamaldehyde or vanillin. The mercapto compound refers to a carboxylic acid, amine or alcohol compound containing a terminal mercapto group, preferably mercaptopropionic acid, mercaptoethanol, mercaptoglycolic acid, cysteine ​​or cysteamine.

[0012] (3) Synthesis of dual-response hyaluronic acid membrane intermediate HA-Tx by esterification reaction: Sodium hyaluronate is pre-prepared into a reaction solution of 0.5%-5% in a mixed solvent, and Tx prepared in step (2) is added dropwise, wherein the ratio of HA to Tx is 1:0.4-1:10, the reaction is heated for 12-48h, dialyzed, and lyophilized; the molecular weight of sodium hyaluronate is 5kDa~500kDa;

[0013] (4) Synthesis of dual-response hyaluronic acid membrane material HA-Tx-xEpo by epoxy ring-opening reaction: Dissolve the product after freeze-drying in step (3) in a mixed solvent, add xEpo prepared in step (1) and alkaline catalyst to the mixed solvent, wherein HA-Tx:xEpo = 1:0.4-1:10, continue heating reaction for 12-48h, precipitate product with alcohol solvent, wash filter cake, dialyze to remove impurities, freeze-dry to obtain final product membrane material HA-Tx-xEpo;

[0014] The mixed solvent used in steps (3) and (4) is a mixture of deionized water and one or two organic solvents selected from N,N-dimethylformamide, dimethyl sulfoxide, acetonitrile, formamide, hexamethylphosphoramide, tetrahydrofuran, dioxane, and N-methylmorpholine.

[0015] In the above technical solution, further, in steps (1) and (4), the alkaline catalyst is one of dimethylaniline, trimethylamine, triethylamine, triethanolamine, pyridine, N-methylpyridine, N-pyrrolidinylpyridine, sodium hydroxide, potassium hydroxide, cesium carbonate, potassium carbonate or sodium carbonate.

[0016] In the above technical solution, the ratio of deionized water to organic solvent in the mixed solvent is 1:0.5-1:20.

[0017] In the above technical solution, further, in step (3), the pre-activated Tx is an active intermediate formed by Tx and one of the following: dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxythiosuccinimide, O-benzotriazole-tetramethylurea hexafluorophosphate, 1,8-diazabicyclo[5.4.0]undec-7-ene, and O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroboronic acid.

[0018] In the above technical solution, the membrane material and the post-genetic agent are further prepared by emulsification solvent evaporation method, solvent diffusion method or high pressure emulsification method to obtain solid lipid nanoparticles (NPs) of the post-genetic agent encapsulated in the membrane material; preferably, the membrane material and the post-genetic agent are mixed with an auxiliary lipid, preferably one or two of stearic acid, palmitic acid, myristic acid, behenic acid, arachidic acid, ceric acid, erucic acid and their mono-, di-, tri- and mixed glycerides.

[0019] In the above technical solution, NPs and prebiotics are further processed by phase separation, spray drying, electrostatic spray freeze drying, sharp-pore coagulation or microfluidic technology to prepare enteric-coated microcapsules (MPs) loaded with NPs and prebiotics. Preferably, the prebiotics and enteric material are mixed as the shell material. Preferably, the enteric material is one or two of cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, acrylic resins II and III, calcium alginate, gelatin and shellac.

[0020] In the above technical solution, taking the preparation of solid lipid nanoparticles (NPs) by emulsification solvent evaporation method and the preparation of microcapsules (MPs) by microfluidic technology as examples: (1) HA-Tx-xEpo, auxiliary lipids and post-biotic drugs (Bas or IPAs) are weighed quantitatively and heated to prepare an oil phase mixed solution for later use; a quantitative oil phase is taken with a dropper and slowly added to an aqueous dispersion containing surfactants, heated and stirred for pre-emulsification, then sonicated with a probe while hot and cooled naturally; dialysis is performed to remove free drugs, thus obtaining a drug-loaded NPs suspension; (2) the NPs suspension is uniformly mixed with a pre-prepared solution containing a quantitative amount of sodium alginate (SA) and prebiotics (xylan, inulin and chitosan oligosaccharide, etc.) to obtain a dispersion, and then dripped into a coagulation bath for solidification by a constant flow pump (the dispersion is injected into the inner needle and an inert gas is introduced into the outer needle), thus obtaining NPs-loaded microcapsules (MPs).

[0021] The present invention also provides the application of the aforementioned drug delivery system in the preparation of oral drug delivery carriers and drugs for chronic intestinal diseases.

[0022] This invention couples SCFAs with hyaluronic acid to obtain the membrane material HA-Tx-xEpo, overcoming the problems of SCFAs' volatility and leakage. It uses ROS-responsive thioacetate / aldehyde linkers and esterase-responsive glyceryl ester bonds, adopting a dual-sensitive response to inflammatory reactions to break the bonds. This allows for better utilization of ROS and glyceryl esterase secreted by inflammatory cells, enabling them to fully respond to the cleavage and release of drugs. Furthermore, the glyceryl ester bond is obtained from the ring-opening reaction of glycidyl carboxylate, and the epoxy group is more reactive than the carboxyl group, resulting in a higher loading rate of small molecule acids and solving the problem of needing high accumulation concentrations for treatment.

[0023] By utilizing HA-Tx-xEpo and auxiliary lipids to encapsulate and load Bas and IPAs, the high boiling points and poor solubility of Bas and IPAs were overcome. Further, inner-layer solid lipid nanoparticles (NPs) were constructed to achieve the release of three drugs at the site of inflammation. Furthermore, the NPs and prebiotics were prepared into enteric-coated microcapsules, achieving a sequential multi-level targeting technology from organ (intestine) to lesion site (inflammatory microenvironment) to inflammatory cells and pathogens. A schematic diagram of the overall structure preparation and responsive release is shown below. Figure 1 The inner layer of solid lipid nanoparticles is made of hyaluronic acid, which exhibits ROS and glycerol esterase-responsive cleavage properties. The molecular weight of the hyaluronic acid is 5kDa to 500kDa. The molecular structure of this hyaluronic acid membrane is as follows: Figure 2 .

[0024] This invention provides a synthetic route and preparation method for the above-mentioned hyaluronic acid membrane material loaded with SCFAs. Taking the HA-TK-BaEpo hyaluronic acid membrane material with butyric acid as the loading drug and thioacetone as the linker arm as an example, the overall synthetic route is as follows: Figure 3 .

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: The present invention forms NPs by using hyaluronic acid coupled with small molecule acids as membrane material to encapsulate postbiotics, and then encapsulates them with prebiotic wall material to form microcapsules. When used, compared with traditional enteritis treatment drugs, it can achieve: (1) sequential multi-level targeting and regional release of different drugs; the MPs of the present invention do not release drugs in the stomach, and almost do not release drugs in a healthy intestinal environment, but start to release drugs when there is inflammation in the intestinal environment; (2) can repair the four major intestinal barriers at the same time, without having a counterproductive effect on barrier repair; (3) promote the proliferation of intestinal epithelial cells and thus repair the mechanical barrier; improve the abundance of intestinal flora and thus repair the microbial barrier; promote mucus secretion and thus repair the chemical barrier; regulate immune homeostasis and thus repair the immune barrier. Attached Figure Description

[0026] Figure 1 A schematic diagram illustrating the fabrication and responsive release properties of the overall structure of the drug delivery system of this invention;

[0027] Figure 2The molecular structural formula of the hyaluronic acid membrane material prepared by this invention;

[0028] Figure 3 Reaction route;

[0029] Figure 4 Schematic diagrams of the preparation of solid lipid nanoparticles (NPs) by emulsification solvent evaporation method and microcapsules (MPs) by microfluidic technology;

[0030] Figure 5 The 1H NMR spectrum of glycidyl butyrate;

[0031] Figure 6 This is the mass spectrum of glycidyl butyrate;

[0032] Figure 7 The mass spectrum of thioacetone;

[0033] Figure 8 The 1H NMR spectrum of thioacetone;

[0034] Figure 9 The mass spectrum of thiocinnamaldehyde;

[0035] Figure 10 The 1H NMR spectrum of HA-TK-BaEpo;

[0036] Figure 11 The infrared absorption spectrum of HA-TK-BaEpo is shown below.

[0037] Figure 12 The 1H NMR spectra of HA-TK-BaEpo with different degrees of substitution are shown.

[0038] Figure 13 The hydrogen NMR spectrum of HA-Tcp;

[0039] Figure 14 The 1H NMR spectrum of HA-Tcp-BaEpo;

[0040] Figure 15 Digital photographs of NPs with different membrane material ratios;

[0041] Figure 16 The graph shows the particle size distribution and the time stability of NPs after initial screening.

[0042] Figure 17 To observe the NPs before and after release using TEM;

[0043] Figure 18 In the ROS environment, NPs release IPA;

[0044] Figure 19 In a ROS environment, NPs release BA;

[0045] Figure 20 Image of MPs prepared for microfluidic technology and observed under an optical microscope;

[0046] Figure 21 The image shows the results of MPs rupture and NPs release in artificial gastrointestinal fluid, observed in situ using an optical microscope.

[0047] Figure 22 This is a graph showing the results of blood compatibility testing.

[0048] Figure 23 The image shows the results of the cell scratch assay.

[0049] Figure 24 This is a graph showing the results of the antibacterial performance test.

[0050] Figure 25 Photograph of the perianal area;

[0051] Figure 26 Photographs of fecal characteristics;

[0052] Figure 27 Photograph of colon length;

[0053] Figure 28 HE-stained section of colon. Detailed Implementation

[0054] The present invention will be further described below with reference to specific embodiments, but this does not limit the present invention in any way.

[0055] Example 1: Synthesis of Dual-Response Hyaluronic Acid Membrane HA-TK-BaEpo

[0056] (1) Preparation of butyric acid epoxy ester

[0057] (a) Raw material preparation: Add 60 mL of anhydrous ethanol and 15 mL of butyric acid, then add 12.75 mL of 5M NaOH solution dropwise. React at 80 °C for 1 h to obtain sodium butyrate. Dry the product to constant weight for later use. (b) Synthesis of epoxy ester: Add 76.2 g of butyrate to 150 g of freshly distilled epichlorohydrin, heat to 110 °C, add 1 g of dimethylaniline hydrochloride catalyst, and stop the reaction after 12 h. Wash the reaction product three times with water, dry with anhydrous magnesium sulfate, and remove excess epichlorohydrin. The product is the crude ester. Separate the crude ester using silica gel column chromatography. 乙酸乙酯 :V 石油醚 Elution was performed using a mobile phase with a ratio of 1:2. After removing the mobile phase, an epoxy ester was obtained. The product was characterized by 1H NMR and mass spectrometry, as shown in the figures below. Figure 5 and Figure 6 .

[0058] (2) Preparation of the linker arm of thioacetal / aldehyde—thioacetone TK

[0059] 3-Mercaptopropionic acid and acetone were added to a three-necked flask in a molar ratio of 2:1.6 under anhydrous and oxygen-free conditions. The reaction was carried out for 15 hours in an acidic atmosphere composed of anhydrous hydrogen chloride. Residual reactants were washed away to obtain the crude product. 乙酸乙酯 :V 石油醚 =1:15 as mobile phase for column chromatography separation. After removing the mobile phase, TK was obtained. The mass spectrum and proton NMR spectrum of the product are shown in the figures below. Figure 7 and Figure 8 .

[0060] (3) Synthesis of dual-responsive hyaluronic acid membrane material HA-TK-BaEpo

[0061] 1.02g of sodium hyaluronate was placed in a mixed solvent (V 甲酰胺 :V 水 Dissolve the product in a 1:2 mixture to prepare a 2% solution. Add 0.68 g of pre-activated TK (molar ratio 1:2) dropwise and heat for 18 h. Dialyze to remove small molecule impurities, freeze-dry, and dissolve again in the mixed solvent for use as a starting material in the next reaction. Add BaEpo and 0.48 g of potassium carbonate to the above mixed solvent and heat for 24 h. Precipitate the product with n-butanol, wash the filter cake, dialyze to remove impurities, and freeze-dry to obtain the final product. The 1H NMR and IR spectra of the product are shown below. Figure 10 and attached Figure 11 Furthermore, by adjusting the feed ratio of HA-TK to BaEpo to 1:0.5, 1:3, 1:6, and 1:9, HA-TK-BaEpo with different degrees of BA substitution was obtained. Figure 12 In the figure, 1, 2, 3, and 4 correspond to the 1H NMR spectra of the products with different feed ratios. When the feed ratio of HA-TK to BaEpo is controlled at 1:3, the degree of butyric acid substitution is 39.8±2.6%, and when the feed ratio is 1:4, the degree of butyric acid substitution is 42.2±2.1%. Taking all factors into consideration, the hyaluronic acid membrane material with a feed ratio of 1:3 was selected for subsequent preparation and therapeutic effect evaluation experiments. Figure 12 The 1H NMR spectra of products with different degrees of BA substitution are shown.

[0062] Example 2: Synthesis of Dual-Response Hyaluronic Acid Membrane HA-Tcp-HaEpo

[0063] (1) Preparation of hexanoic acid epoxy ester

[0064] (a) Raw material preparation: Add 50 mL of anhydrous methanol and 12.5 mL of hexanoic acid, then add 10.5 mL of 5M NaOH solution dropwise. React at 80 °C for 1 h to obtain hexanoate. Dry the product to constant weight for later use. (b) Synthesis of epoxy ester: Add 84.4 g of hexanoate to 150 g of freshly distilled epichlorohydrin, heat to 110 °C, add 0.84 g of catalyst 4-pyrrolidinylpyridine, and react for 20 h. Wash the reaction product three times with water, dry with anhydrous magnesium sulfate, and remove excess epichlorohydrin. The product is the crude ester. Separate the crude ester using silica gel column chromatography. 乙酸乙酯 :V 石油醚 A gradient elution was performed using a mobile phase with a ratio of 1:5, and the epoxy ester was obtained after removing the mobile phase.

[0065] (2) Preparation of the linker arm of thioketal / aldehyde—thiocinnamaldehyde Tcp

[0066] 0.05 mol of 3-mercaptopropionic acid and 0.015 mol of cinnamaldehyde were added to a three-necked flask under anhydrous and oxygen-free conditions. The reaction proceeded overnight in an acidic atmosphere composed of zinc chloride, involving the addition of the thiol group to the carbonyl group. Residual reactants were washed away to obtain the crude product. (The text abruptly ends here, so the translation stops as well.) 乙酸乙酯 :V 石油醚 A mobile phase ratio of 1:8 was used. After removing the mobile phase, Tcp was obtained. The high-resolution mass spectra of the product are shown in [reference needed]. Figure 9 .

[0067] (3) Synthesis of dual-responsive hyaluronic acid membrane material HA-Tcp-HaEpo

[0068] Sodium hyaluronate was dissolved in a mixed solvent V. 甲酰胺 :V 水 In a 10:3 mixture, an appropriate amount of pre-activated Tcp was added dropwise, and the reaction was heated for 24 hours. After dialyzing and lyophilization, the product was dissolved again and used as a starting material for the next reaction. HaEpo and an appropriate amount of catalyst were added to the above mixed solvent, and the reaction was heated for 32 hours. The product was precipitated with isopropanol, the filter cake was washed, impurities were removed by dialyzing, and the final product was obtained by lyophilization.

[0069] Example 3: Preparation of solid lipid nanoparticles (NPs)

[0070] 500.0 mg of the membrane material HA-TK-Ba (HA-TK to BaEpo feed ratio 1:3), 324 mg of auxiliary lipid (glyceryl stearate monoester MG), and 37.5 mg of post-biotic drug (indolepropionic acid IPA) were quantitatively weighed in Example 1 and heated and stirred to prepare an oil phase mixture solution for later use. A quantitative amount of the oil phase was drawn up using a dropper and slowly added to 100 mL of an aqueous solution containing 0.5% Tween-80 for emulsification. After natural cooling, the NPs solution was transferred to a dialysis bag to remove free drug, thus obtaining a drug-loaded NPs suspension. The effect of different membrane material ratios on the particle size distribution and time stability of NPs was investigated. Figure 15 , 16 The membrane material ratio was determined to be 8:5 (i.e., HA-TK-Ba:MG = 8:5), and subsequent series of experiments were conducted based on this ratio.

[0071] Example 4: In vitro drug release from solid lipid nanoparticles (NPs)

[0072] Using 50 μM H2O2 and 5 μM Fe 2+ Aqueous solutions underwent a Fenton reaction to generate ROS, and 15 μM lipase was added to create a simulated body fluid to mimic the inflamed intestinal microenvironment. PBS was used as a control group to simulate the healthy body fluid microenvironment. NPs prepared in Example 3 (HA-TK-Ba:MG = 8:5) were added to both simulated body fluids, and timing was started. Morphological changes before and after the response release were observed using transmission electron microscopy at 0 h and 12 h. Figure 17 ),Depend on Figure 17 The results showed that in a healthy microenvironment, NPs remained almost intact spheres. However, under simulated inflammatory microenvironment stimulation, NPs changed from intact spheres to loose, irregular fragments, indicating that NPs can structurally break down and disintegrate under inflammatory conditions. The release of IPA from the encapsulated contents was observed using simulated body fluids collected from 0 to 24 hours. Since IPA has ultraviolet absorption, ultraviolet thin-layer chromatography was used to observe the release of IPA. Figure 18 The study found that IPA was gradually released in the simulated inflammatory intestinal microenvironment and, in the simulated inflammatory body fluids, was gradually released over time. Figure 18 It can be seen that IPA release begins around 6 hours; the release of butyrate (BA) was determined by gas chromatography using simulated body fluid samples taken from 0-48 hours. Figure 19 Therefore, NPs have the effect of releasing drugs in response to inflammation.

[0073] Example 5: Preparation of prebiotic microcapsules (MPs)

[0074] Microcapsules were prepared using gas-liquid shear microfluidics. The NPs suspension from Example 3 was mixed in an electromagnetic stirrer with an equal volume of a pre-prepared solution of 4% sodium alginate (SA) and prebiotics (2.4% xylooligosaccharides and 1.6% inulin) to obtain a dispersion. A quantitative amount of the dispersion was injected using a syringe and passed through a constant flow pump (dispersion injection into the inner needle at a flow rate of 800 μL / min, and inert gas N2 or CO2 introduced into the outer needle at a flow rate of 4 L / min). Under the combined action of gas shear force and gravity, the dispersion was dripped into a coagulation bath (containing an aqueous solution of 0.5% chitosan oligosaccharide and 0.5% calcium chloride) for solidification, ultimately yielding NPs and prebiotic-loaded microcapsules (MPs). A schematic diagram of the apparatus for preparing MPs using gas-liquid shear microfluidics is shown below. Figure 4 ) and observation under a stereomicroscope Figure 20The obtained MPs are uniform spherical particles with a particle size of about 100 μm; the microcapsules are golden yellow and translucent, in which the NPs loaded inside give the microcapsules their translucent characteristics, while golden yellow is the characteristic color of chitosan oligosaccharides.

[0075] Example 6: Prebiotic microcapsules (MPs) for in vitro drug release

[0076] The pH-responsive release properties of the MPs were investigated. The MPs from Example 5 were used in an in vitro drug release experiment in simulated gastric fluid (USP version of SGF). The morphology of the MPs did not show significant changes. Figure 21 a; In vitro drug release experiments were conducted using artificial intestinal fluid (USP version of SIF). Morphological images of MPs at different time points were observed and photographed in situ under a stereomicroscope, see... Figure 21 b, in artificial intestinal fluid. This indicates that the drug delivery system of this application only begins to lyse and release the drug after reaching the intestines. Figures 17-19 The results showed that MPs maintained their structural integrity in gastric juice; after rupturing and disintegrating in the intestine, they released prebiotics and low concentrations of postbiotics from NPs, which played a preventive role in maintaining a healthy intestinal microenvironment; when there was inflammatory stimulation in the intestinal lumen, NPs ruptured and released high concentrations of postbiotic drugs to treat and repair the damaged intestinal microenvironment.

[0077] Example 7: In vitro hemolysis experiment of drug delivery system

[0078] Fresh anticoagulated pig blood was centrifuged at low speed, the supernatant was discarded, and the blood was washed with physiological saline. The mixture was centrifuged again until the supernatant was colorless, yielding the erythrocyte stock solution. The erythrocyte suspension was diluted to 2% with physiological saline. Three solutions (S1, S2, and S3) prepared according to concentration gradients of 20, 50, 200, and 500 μg / mL for Postbiotics, NPs, and MPs were added sequentially to the 2% blood cell suspension. The solutions were incubated in a constant temperature water bath, centrifuged, and the supernatant was collected. The absorbance at 416 nm was measured. For 0% hemolysis (physiological saline group) and 100% hemolysis (Trappon group), the stock solution was diluted directly with physiological saline by the same factor, and the absorbance value was measured as the absorbance of the solvent itself as a control. The percentage of hemolysis was calculated according to Formula 1.

[0079]

[0080] After incubation for 2 hours and centrifugation, the morphology of the red blood cell suspension is shown in the following image. Figure 22 It can be seen that the red blood cells in the Triton group were completely broken down, releasing hemoglobin. After centrifugation, the supernatant was light reddish-brown with no red blood cell precipitation. In contrast, samples S1, S2, and S3 did not show significant hemolysis even at a concentration of 500 μg / mL, similar to the saline group. This indicates that the selected drug, the synthesized carrier, and the overall delivery system all have good biocompatibility.

[0081] Example 8 Cell Scratch Test

[0082] Draw parallel lines on the back of a 6-well plate using a marker pen. Digest cells with trypsin to prepare a single-cell suspension. After counting, seed the cells, ensuring a uniform seeding density for each group. Once the cells have confluently formed, use a 20µL pipette tip perpendicular to the wells and lines to create cell scratches. Change the medium and remove the scratched cells. Add simulated inflammation culture medium (containing 50µM H₂O₂ and 5µM Fe₂O₃). 2+ Cells were added with 15 μM lipase, and then the drugs were added. The naked drug groups S1 (BA 10 μM and IPA 6.4 μM), S2, and S3 contained the same mass of drug carriers (S2 contained NPs 0.42 mg / mL and S3 contained MPs 1.03 mg / mL). Cell migration ability under inflammatory conditions was assessed by comparing the scratch area at the initial scratch (0 h) and later observation points (24 h). Grayscale images of the microscopic photographs are shown below. Figure 23 As shown, groups S1, S2, and S3 all have a certain ability to promote cell migration, suggesting that they may have the ability to repair and treat tissue mucosa, laying the foundation for subsequent animal experiments.

[0083] Example 9 Antibacterial properties

[0084] Materials S1, S2, S3, and antibiotic (ABX) of the same mass as in Example 8 were uniformly mixed with nutrient agar at 50-60°C, poured into plates, and allowed to cool completely. 200 μL of the prepared culture was then spread using a bacterial spreader. 6 CFU·mL -1 Fresh *Escherichia coli* and *Staphylococcus aureus* were spread onto agar plates. After incubation at 37°C for 24 hours, the colony counts were observed and recorded. The results are shown in the attached figure. Figure 24 As shown, no visible bacterial colonies grew in groups S1 and ABX, while colonies grew in groups S2 and S3. The number of colonies in S3 was less than that in S2, possibly because the prebiotic chitosan oligosaccharide in MPs also has a certain antibacterial effect. The presence of colonies in S2 and S3 verifies and illustrates that not all bacteria in the gut were eliminated, but only bacteria invading the body at the site of inflammation were eliminated, minimizing damage to the microbial barrier and thus achieving a healthy homeostatic microenvironment in the gut where different bacterial communities mutually restrain and depend on each other.

[0085] Example 10: DSS-induced mouse ulcerative colitis model and evaluation of drug treatment efficacy.

[0086] Three days before modeling, drugs were administered. The DSS+API and DSS+MPs groups received the same volume of drug via gavage at a fixed time each morning; the DSS group received the same volume of PBS solution via gavage. A DSS-induced ulcerative colitis (UC) model was established in C57BL6 mice, lasting for 7 days. Fresh 2.5% DSS deionized water was provided for free drinking daily. Mouse weight, fecal matter, and perianal condition were recorded. Seven days after modeling, mice were sacrificed, colonic tissue was collected, and photographs were taken. Perianal and fecal characteristics were analyzed... Figure 25-26 ), colon length ( Figure 27 ) and HE-stained sections of colon ( Figure 28 This study aims to preliminarily investigate the therapeutic efficacy of a sequential multi-stage targeted drug delivery system in UC. Figure 25-26 The results showed that the DSS group had fecal matter adhering to the perianal area, with obvious visible blood in the stool. After the model treatment, fresh colon tissue was dissected, and the colon length and pathological sections were observed and recorded for analysis. Figure 27 Compared to the DSS group, the colon length of both the NPs and MPs treatment groups increased significantly, with the MPs group showing the longest colon length. This indicates that MPs can significantly inhibit apoptosis and necrosis of intestinal epithelial cells caused by ulcerative colitis and promote epithelial cell proliferation and differentiation. The combination of MPs' prebiotic wall material and postbiotic further enhanced the treatment effect. Figure 28 HE staining of colonic sections showed that the colonic crypts in the DSS group were almost completely disappeared, with a large number of inflammatory cells aggregated. Compared with the NPs group, the colonic crypt structure in the MPs group was more intact and there was less inflammatory cell infiltration. Therefore, the treatment with the MPs system is appropriate and effective.

[0087] For anyone skilled in the art, many possible variations and modifications can be made to the technical solutions of this invention, or equivalent embodiments can be modified based on the disclosed technical content, without departing from the scope of the technical solutions of this invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of this invention without departing from the content of the technical solutions of this invention should still fall within the protection scope of the technical solutions of this invention.

Claims

1. A sequential multi-stage targeted drug delivery system for repairing the intestinal barrier, characterized in that, The delivery system is microcapsules (MPs) encapsulating NPs; the NPs are solid lipid nanoparticles formed by encapsulating postbiotics with hyaluronic acid coupled with small molecule acids as membrane material, and the postbiotic is indolepropionic acid (IPA); the shell material of the microcapsule MPs is a prebiotic, and the prebiotic is xylooligosaccharide and inulin. The method for preparing the membrane material includes the following steps: (1) Dehydration cyclization synthesis of glycidyl oxide xEpo: A small molecule acid is reacted with an alkaline solution to obtain a small molecule acid salt, which is added to freshly distilled epichlorohydrin. An alkaline catalyst is added and the mixture is heated to reflux. After reacting for 2-24 hours, the reaction is stopped. Excess epichlorohydrin is removed from the reaction product, and xEpo is obtained by column chromatography. The small molecule acid is butyric acid or hexanoic acid. (2) Synthesis of thioacetal / ketone Tx by carbonyl addition reaction: The mercapto compound and the natural aldehyde / ketone compound are reacted at a molar ratio of 2:1-2:2.5 under an acidic atmosphere for 12-48 h. The residual reactants are washed off and purified by column chromatography to obtain the product Tx. The mercapto compound is 3-mercaptopropionic acid. The natural aldehyde / ketone compound is acetone or cinnamaldehyde. (3) Synthesis of dual-response hyaluronic acid membrane intermediate HA-Tx by esterification reaction: Sodium hyaluronate HA is pre-prepared into a reaction solution of 0.5%-5% in a mixed solvent, and pre-activated Tx is added dropwise, wherein the ratio of HA to Tx is 1:0.4-1:

10. The reaction is heated for 12-48h, dialyzed, and lyophilized to obtain HA-Tx; (4) Synthesis of dual-response hyaluronic acid membrane material HA-Tx-xEpo by epoxy ring-opening reaction: Dissolve lyophilized HA-Tx in a mixed solvent, add xEpo and alkaline catalyst, wherein HA-Tx:xEpo = 1:0.4-1:10, continue heating reaction for 12-48h, precipitate product with alcohol solvent, wash filter cake, dialyze to remove impurities, and lyophilize to obtain product membrane material HA-Tx-xEpo; The alkaline catalysts in steps (1) and (4) are dimethylaniline hydrochloride, potassium carbonate, or 4-pyrrolidinylpyridine; the mixed solvents in steps (3) and (4) are a mixture of deionized water and N,N-dimethylformamide in a ratio of 1:0.5-1:

20.

2. The sequential multi-stage targeted drug delivery system for repairing the intestinal barrier according to claim 1, characterized in that, Enteric-coated microcapsules (MPs) loaded with NPs and prebiotics were prepared by phase separation, spray drying, electrostatic spray freeze drying, sharp-pore solidification, or microfluidic technology.

3. The drug delivery system according to any one of claims 1 to 2 is used in the preparation of oral drug delivery carriers and drugs for chronic intestinal diseases.

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