A probiotic delivery system based on a core-shell hydrogel and its application

Abstract

This invention discloses a probiotic delivery system based on a core-shell hydrogel structure and its application, belonging to the field of biomedical technology. The system comprises a hydrogel capsule shell and a core containing probiotics. The hydrogel capsule shell isolates the probiotics from the external environment and adsorbs lipopolysaccharides produced by the probiotics. The internal pore size of the capsule shell is 200-300 nm, allowing external nutrients to enter and the release of therapeutically active metabolites secreted by the probiotics. The core is a sodium alginate hydrogel that effectively maintains bacterial activity and contains certain nutrients required by the bacteria. This probiotic delivery system provides dual isolation effects, combining physical and chemical isolation, avoiding the negative effects of bacterial endotoxins on the human body. It achieves a balance between bacterial isolation and drug penetration, exhibiting good long-term stability.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a probiotic delivery system based on a core-shell hydrogel and its application. Background Technology

[0002] Drug depot systems, as a mature strategy for local drug delivery, play a crucial role in the management of chronic diseases, including inflammatory diseases, endocrine disorders, and malignancies. These systems generally include injectable controlled-release formulations and surgically implanted devices, enabling sustained release of therapeutic drugs, thereby reducing systemic exposure and improving target site specificity. Reduced dosing frequency helps maintain stable circulating drug concentrations and improves patient adherence. However, traditional drug depot systems have limitations in terms of total drug release; they lose their effectiveness once the drug load is depleted and cannot be refilled non-invasively. These problems are particularly pronounced when the implantation site is difficult to access.

[0003] To address the limitations of drug depot dosage and the difficulty of refilling, the concept of in vivo drug depots (LDDs) has emerged. These depots utilize living cells, rather than pre-prepared drugs, to achieve continuous therapeutic drug production. These cells can achieve customized and controlled drug delivery by secreting specific therapeutic molecules. The living cells encapsulated within the LDD can autonomously extract nutrients from the surrounding microenvironment, proliferate, and locally synthesize and secrete therapeutic molecules. This allows for continuous drug production without external supplementation. Among the various cell types explored, bacteria possess unique advantages due to their ease of cultivation, rapid proliferation rate, and relatively low nutritional requirements, making them a particularly attractive candidate for in vivo therapeutic modalities.

[0004] Despite the increasing prevalence of live bacteria in therapeutic applications, the development of safe and stable drug repositories based on live bacteria remains a significant challenge, with little progress yet to be made. This is because the safe and effective use of live bacteria in the human body requires both physical and chemical isolation. Physically, bacteria must be confined to prevent uncontrolled proliferation or premature clearance by the host's immune system before therapeutic effects are achieved. Chemically, endotoxins released during bacterial lysis must be neutralized, as these endotoxins can act as nonspecific immunogens, triggering adverse symptoms such as fever. Furthermore, the implantable carriers used to hold the bacteria must possess sufficient mechanical strength to prevent bacterial leakage and ensure safe and complete removal after treatment. This is crucial for reducing the risk of uncontrolled bacterial spread or disruption of the local microbiome.

[0005] Chinese patent document CN119223942A discloses a magnetic hydrogel-encapsulated bacterial biosensor platform. This invention uses sodium alginate hydrogel to encapsulate bacterial strains and magnetic Fe3O4 microparticles to form a biosensor for detecting intestinal inflammation. The bacterial strains report the presence of the inflammatory marker heme through bioluminescent signal expression, while the presence of magnetic Fe3O4 microparticles allows for efficient recovery of intact microspheres from feces via magnetic adsorption. This provides a theoretical basis and new method for achieving efficient, rapid, and accurate in vitro detection and diagnosis of intestinal inflammation. Chinese patent document CN119632947A discloses a leak-free core-shell hydrogel microsphere delivery system for non-contact microbiota transplantation. This invention encapsulates bacteria within the core of core-shell microspheres using a two-step photoinitiated emulsion polymerization method with methacrylated gelatin, constructing a non-contact microbiota transplantation system that demonstrates good therapeutic efficacy and safety.

[0006] However, the existing bacterial delivery systems have the following drawbacks: (1) Core-shell hydrogels are mostly delivered in the form of microspheres. Although this form is convenient for oral administration due to its small size, its bulk form (usually oral administration or implantation of multiple microspheres) poses a challenge to complete recovery after treatment (if bacteria remain in the body for a long time, they may affect the original flora or cause infection due to uncontrolled proliferation or potential gene mutations), and often limits the integration of additional functions; (2) A major pain point limiting the application of live bacteria in vivo is the uncontrolled proliferation and spread of bacteria. The toxins and certain metabolites produced by bacteria can induce infection. This is particularly fatal in the case of subcutaneous bacterial implantation, which directly hinders the development of subcutaneous implantation systems carrying live bacteria. Many existing live bacterial delivery systems ignore the adverse effects of lipopolysaccharide (LPS) produced by bacterial lysis on the human body. Bacterial LPS can induce systemic inflammation through TLR4-mediated cytokine storms. (3) Existing sodium alginate-acrylamide hydrogels have limited mechanical strength due to swelling in the solution environment. If used in the in vivo environment, they will be difficult to maintain structural integrity, which may cause bacteria to leak and proliferate uncontrollably in the body, bringing a series of negative effects. Summary of the Invention

[0007] To address the shortcomings of the existing technologies, this invention provides a hydrogel capsule shell for probiotic delivery and a probiotic delivery system based on a core-shell hydrogel structure. The hydrogel capsule shell provides a stronger and more stable physical isolation effect, preventing probiotic (bacterial) leakage and preventing inflammation caused by bacterial lipopolysaccharides. The probiotic delivery system based on a core-shell hydrogel structure has broad application prospects in the field of LDD (Liquid Degradation).

[0008] The specific technical solution adopted is as follows:

[0009] A hydrogel capsule shell for probiotic delivery, the preparation method of which includes the following steps:

[0010] A hydrogel precursor solution for the capsule shell was prepared using sodium alginate, acrylamide, a selective lipopolysaccharide remover, an initiator, and a first crosslinking agent. An accelerator was then added to induce the hydrogel to form a preliminary gel, thus preparing a capsule shell precursor. The capsule shell precursor was then subjected to multiple crosslinking processes in MES buffer containing a second crosslinking agent, an activator, and an activation enhancer, as well as in a solution containing calcium ions, to obtain the hydrogel capsule shell.

[0011] The outer shell of the hydrogel capsule is a sealed shell, and the hollow part inside is used to load probiotics to isolate the probiotics from the outside world.

[0012] The first crosslinking agent is selected from N,N'-methylenebisacrylamide or N,N'-bisacrylpiperazine; the second crosslinking agent is selected from adipate dihydrazide.

[0013] The initiator is selected from ammonium persulfate or potassium persulfate;

[0014] The accelerator mentioned is N,N,N',N'-tetramethylethylenediamine;

[0015] The activator is selected as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide;

[0016] The activating and enhancing agent is N-hydroxysuccinimide.

[0017] The hydrogel capsule shell for probiotic delivery provided by this invention is a sodium alginate-polyacrylamide hydrogel incorporating LPS selective removal agent. Due to the multiple cross-linking steps, the hydrogel has significantly improved mechanical properties and anti-swelling ability, which can play a higher strength and more stable physical isolation role, prevent probiotic (bacterial) leakage, and prevent bacterial lipopolysaccharide-induced inflammation.

[0018] Furthermore, the lipopolysaccharide selective removal agent is an amino acid or a polycationic substance. The amino acid includes histidine, lysine, or arginine, and the polycationic substance includes polyethyleneimine (PEI), poly-L-lysine (PLL), or poly-L-histidine (PLH).

[0019] Preferably, in the shell hydrogel precursor solution, the solvent is water, the mass fraction of sodium alginate is 1-3 wt%, the mass fraction of acrylamide is 10-30 wt%, the mass fraction of the first crosslinking agent is 0.05-0.08 wt%, the content of the initiator is 0.005-0.01 wt%, the concentration of the lipopolysaccharide selective removal agent is 1-10 mM, and the amount of accelerator added is 0.01-0.1 wt% of the shell hydrogel precursor solution.

[0020] After the accelerator is added, the shell hydrogel precursor solution will quickly solidify to form the capsule shell precursor.

[0021] The most preferred configuration is that the mass fraction of sodium alginate in the hydrogel precursor solution is 2 wt%, and the mass fraction of acrylamide is 20 wt%. Experiments have shown that the hydrogel capsule shell prepared under these parameters exhibits the best performance.

[0022] Preferably, in the MES buffer containing a second crosslinking agent, an activator, and an activation enhancer, the concentration of the activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 5-8 mM, the concentration of the activation enhancer N-hydroxysuccinimide is 1.5-5 mM, and the concentration of the second crosslinking agent adipate dihydrazide is 5-15 mM.

[0023] Preferably, the capsule shell precursor is placed in a MES buffer solution containing a second crosslinking agent, an activator, and an activation enhancer for crosslinking at 0-40°C for 0.5-1 h. After removal, it is placed in a solution containing calcium ions for crosslinking at 0-40°C for 0.5-1 h to obtain the hydrogel capsule shell.

[0024] Further preferably, the solution containing calcium ions is a CaCl2 solution with a concentration of 0.5-1.0 M.

[0025] Preferably, the hydrogel capsule shell has an internal pore size of 200-300 nm, which can adsorb lipopolysaccharides produced by probiotics, allowing external nutrients to enter and probiotics to release therapeutic metabolites.

[0026] The present invention also provides a probiotic delivery system based on a core-shell hydrogel, the structure of which includes the aforementioned hydrogel capsule shell and a core containing probiotics;

[0027] The method for preparing the probiotic-containing core includes: mixing sodium alginate solution and probiotic culture medium to obtain a core precursor solution; transferring the core precursor solution to the hollow part of the hydrogel capsule shell; allowing it to stand so that calcium ions in the hydrogel capsule shell can permeate into the core precursor solution and crosslink to form a core, thereby obtaining the probiotic delivery system based on the core-shell structure hydrogel.

[0028] The probiotics mentioned are live bacteria (with or without gene editing) that can secrete metabolites with therapeutic effects.

[0029] The aforementioned probiotic delivery system based on a core-shell hydrogel structure serves as an enhanced LDD platform. Through an optimized delivery system (this invention proposes a method of integrally encapsulating live bacteria in capsule-like / rod-shaped core-shell hydrogels (“macro-encapsulation”), which facilitates complete removal after treatment and has the potential to integrate auxiliary technologies such as drug delivery devices), it encapsulates microorganisms, adsorbs lipopolysaccharides produced by the bacteria, and ensures continuous therapeutic secretion while minimizing immune responses. In animal models of tumors and radiation enteritis, implantation and endoscopic drug delivery of LDDs carrying different bacteria demonstrated sustained release, higher efficacy, and long-term safety. This invention's delivery system provides a multifunctional and effective solution for long-term local drug delivery, overcoming key obstacles in live bacterial therapy.

[0030] Preferably, the sodium alginate solution and probiotic culture medium are mixed in an equal volume ratio, and the OD of the probiotic culture medium is... 600 >0.2 indicates that the probiotic culture medium contains carbon sources, nitrogen sources, growth factors, inorganic salts, or osmotic pressure regulators that can be utilized by probiotics.

[0031] More preferably, the probiotics are Bifidobacterium, Lactobacillus, Lactococcus lactis, Lactobacillus acidophilus, etc. (which can secrete short-chain fatty acids, bacteriocins, etc., which have the effect of inhibiting pathogenic bacteria), or non-pathogenic Escherichia coli that can secrete immune checkpoint inhibitors (PD-L1 nanobodies, CTLA-4 nanobodies, etc.) and neutralizing antibodies against pro-inflammatory factors (NF-α and IL-23 bispecific antibodies, etc.) through gene editing.

[0032] Preferably, the core precursor solution is injected into the hollow portion of the hydrogel capsule shell obtained in step (1).

[0033] Preferably, the raw materials for the hydrogel capsule shell include acrylamide, sodium alginate, and histidine. Acrylamide is used as a monomer, ammonium persulfate as an initiator, and N,N'-methylenebisacrylamide as a crosslinking agent to prepare a polyacrylamide hydrogel. Then, polyacrylamide and sodium alginate form covalent bonds in a MES buffer containing a crosslinking agent and a catalyst. Further soaking in 1.0 M CaCl2 allows the sodium alginate to crosslink under calcium ion-mediated crosslinking, resulting in the final hydrogel capsule shell.

[0034] Preferably, the core is composed of a hydrogel precursor made by mixing sodium alginate and bacterial culture, and then passing it through Ca... 2+ The process mediates cross-linking of the core, forming a sodium alginate hydrogel that can better maintain bacterial activity and contains certain nutrients required by the bacteria.

[0035] Optionally, the probiotic delivery system based on core-shell hydrogel is cylindrical with a diameter of no more than 15 mm and a height of no more than 30 mm.

[0036] The present invention also provides a drug residence structure, including the probiotic delivery system and residence component based on the core-shell structure hydrogel.

[0037] Furthermore, the drug-residue structure can deliver probiotics to the skin, subcutaneous tissue, gastrointestinal tract, urogenital tract, and other sites for the treatment of tumors and various inflammatory diseases, including radiation colitis.

[0038] Furthermore, the residency component is used to protect the probiotic delivery system based on the core-shell structure hydrogel and fix it to the intestinal wall.

[0039] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0040] (1) The probiotic delivery system based on core-shell hydrogel provided by the present invention has dual isolation effects of physical isolation and chemical isolation: physical isolation of bacteria: while isolating bacteria inside, the outer shell of the hydrogel capsule allows external nutrients and internal secretion products (such as PD-L1 nanobodies, TNF-α and IL-23 bispecific antibodies, GLP-1, etc.) to enter and exit, thus achieving the effect of a local reservoir for continuously secreted drugs; chemical isolation of LPS: histidine and other substances are incorporated into the hydrogel shell to neutralize and adsorb LPS, thereby avoiding the negative effects of bacterial endotoxins on the human body (especially when applied in subcutaneous implantation scenarios).

[0041] (2) The probiotic delivery system based on core-shell hydrogel provided by the present invention is a combination design of sodium alginate hydrogel core and sodium alginate-polyacrylamide multi-crosslinked shell layer to achieve a balance between bacterial isolation and drug penetration. Among them, the sodium alginate-polyacrylamide outer layer has the characteristics of multiple crosslinking, including crosslinking between sodium alginate and polyacrylamide, crosslinking inside polyacrylamide, and calcium ion-mediated crosslinking of sodium alginate, etc., thereby achieving long-term stability of the system in simulated gastrointestinal scenarios. Attached Figure Description

[0042] Figure 1 The images show the preparation process of the probiotic delivery system based on a core-shell hydrogel and real-life photos of different sizes, with Escherichia coli Nissle1917 selected as the probiotic.

[0043] Figure 2 The effect of different mass ratios of sodium alginate and acrylamide in the preparation of hydrogel capsule shells.

[0044] Figure 3 The image shows a comparison of the swelling properties of the hydrogel capsule shell before and after the calcium ion crosslinking step. SGF represents simulated gastric juice (pH 1.5, containing enzymes), and SIF represents simulated intestinal juice (pH 7.0, containing enzymes).

[0045] Figure 4 Cryo-electron microscopy images and pore area statistics of a probiotic delivery system based on a core-shell hydrogel structure.

[0046] Figure 5 A comparison of the mechanical properties of the hydrogel capsule shell before and after the calcium ion crosslinking step (cyclic compression test).

[0047] Figure 6 The graph shows the adsorption effect of bacterial LPS on the probiotic delivery system based on core-shell hydrogel. Gel represents the group loaded with bacteria using a hydrogel capsule shell without histidine, and His-Gel represents the group loaded with bacteria using a hydrogel capsule shell containing different concentrations of histidine.

[0048] Figure 7 The therapeutic effects on tumors after subcutaneous implantation of the probiotic delivery system based on the core-shell hydrogel structure in mice were evaluated (Group 1: no treatment; Group 2: implantation of blank hydrogel capsule shell; Group 3: tail vein injection of engineered bacteria; Group 4: implantation of the probiotic delivery system based on the core-shell hydrogel structure).

[0049] Figure 8 The images show actual photos of the drug retention components and the drug retention structure containing the probiotic delivery system based on the core-shell hydrogel. Detailed Implementation

[0050] The present invention will be further illustrated below with reference to the embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0051] Example 1: Preparation of hydrogel capsule shell for probiotic delivery

[0052] (1) Prepare an aqueous solution containing 2 wt% sodium alginate and 20 wt% acrylamide. After dissolving completely, add histidine to make the final concentration in the shell hydrogel precursor solution 1 mM. Then add 0.01 wt% ammonium persulfate and 0.05 wt% N,N'-methylenebisacrylamide, mix thoroughly, remove bubbles, and obtain the shell hydrogel precursor solution. Pour the shell hydrogel precursor solution after removing bubbles into a mold, add 0.05% (vt / vt) of gel accelerator N,N,N',N'-tetramethylethylenediamine (TEMED), and wait at room temperature for less than 15 minutes to complete the initial gelation (acrylamide polymerization) to prepare the capsule shell precursor. Then immerse the capsule shell precursor in MES buffer (0.1 MMES and 0.5 M NaCl, pH 6.0) containing a second crosslinking agent, activator and activation enhancer for crosslinking at room temperature for 45 minutes. The MES buffer containing the second crosslinking agent, activator and activation enhancer contains 8 The hydrogel capsule shell is composed of 1 mM activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 5 mM activator enhancer N-hydroxysuccinimide (NHS), and 10 mM second crosslinking agent adipate dihydrazide (ADH), which forms covalent bonds between sodium alginate and the polyacrylamide network. To further optimize the performance of the hydrogel capsule shell, the hydrogel capsule shell is soaked and crosslinked at room temperature for 45 minutes using a 1.0 M CaCl2 solution to obtain the hydrogel capsule shell for probiotic delivery.

[0053] When determining the formulation of the hydrogel capsule shell, the ratio of sodium alginate to acrylamide was screened. We used 0-3 wt% sodium alginate and 5-10 wt% acrylamide, and finally determined that hydrogels containing 2-3 wt% sodium alginate and 20 wt% acrylamide could effectively encapsulate bacteria. Figure 2 ).

[0054] like Figure 3 As shown, the cross-linking step added in this invention greatly reduces the swelling rate of the hydrogel capsule shell in simulated gastrointestinal fluid (by about 50%), which is beneficial for the long-term stable application of the system in the gastrointestinal environment.

[0055] Meanwhile, the added calcium ion crosslinking step significantly increases the mechanical properties of the hydrogel capsule shell, as shown in the compression test results. Figure 5As shown, the sodium alginate-polyacrylamide hydrogel of this invention can withstand a pressure of approximately 30 kPa when deformed by 60%. Based on the pressure range within the gastrointestinal tract: esophagus: 4-8 kPa (peak during swallowing); stomach: 0.5-2 kPa (basal peristalsis), 3-10 kPa (during strong contractions or vomiting); small intestine: 1-5 kPa (segmental contractions and propulsive peristalsis); colon: 2-15 kPa (high-amplitude propagating contractions, such as during defecation). Therefore, the outer shell of this hydrogel capsule can fully withstand the pressure at various locations within the digestive tract, which is beneficial for the application of the system in environments such as the gastrointestinal tract. Furthermore, the mechanical properties of this hydrogel system also make it suitable for applications such as implantation and wearable devices.

[0056] Example 2: Probiotic Delivery System Based on Core-Shell Hydrogel Structure

[0057] (1) Genetically edited bacteria can produce immune checkpoint inhibitors, such as PD-L1 nanobodies and CTLA-4 nanobodies (used in tumor treatment).

[0058] Plasmid design: After confirming the binding of the nanobodies to their respective targets, the sequences of the PD-L1 and CTLA-4 nanobodies were cloned into a high-copy plasmid for strong constitutional typing. tac To achieve maximum gene expression, a separate plasmid downstream of the promoter was used. A human influenza hemagglutinin (HA) protein tag was added to the 3' end of the nanobody sequence for in vitro visualization, and the Axe / Txe stabilization mechanism was cloned into a vector to prevent plasmid loss during bacterial replication; for details, please refer to the literature Candice R. Gurbatri et al . Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies. Sci. Transl. Med .12, eaax0876(2020). DOI: 10.1126 / scitranslmed.aax0876 Genetically engineered bacteria that secrete PD-L1 and CTLA-4 nanobodies.

[0059] (2) Genetically edited bacteria can produce neutralizing antibodies against pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α) neutralizing nanobodies (targeting inflammation).

[0060] Editing Methods: Non-pathogenic *E. coli* (EcN or HS strains) were engineered using synthetic biology techniques: First, the pathogenic IPA operon of the Shigella type III secretion system (T3SS) was deleted, retaining only the mxi-spa structural gene cluster, which was then integrated into a specific chromosomal site, transforming T3SS from a host cell injection mode to an environmental secretion mode. Second, the constitutive promoter PJ23119 was used to drive the expression of the transcriptional regulator VirB, achieving continuous activation of the secretion system. Subsequently, the OspC2 secretion signal peptide was fused to the N-terminus of a therapeutic nanobody (such as anti-TNF-α) to ensure efficient secretion of the heterologous protein while maintaining its functional activity. Finally, the host's alar / dadX gene was knocked out, and an alar compensation gene was introduced into the expression vector to construct a stable symbiotic system that does not require antibiotic selection, enabling long-term colonization of the engineered bacteria in the gut and continuous secretion of therapeutic proteins. For details, please refer to the literature Jason P. Lynch, Coral González-Prieto, Analise Z. Reeves. et al . EngineeredEscherichia coli for the in situ secretion of therapeutic nanobodies in thegut. Cell Host & Microbe 31, 634-649 (2023). https: / / doi.org / 10.1016 / j.chom.2023.03.007. Preparation of genetically engineered bacteria that secrete tumor necrosis factor-α (TNF-α) neutralizing nanobodies.

[0061] The preparation process of the hydrogel core is as follows: First, a 5 wt% sodium alginate solution and a bacterial culture medium (specifically, Escherichia coli Nissle1917 in Luria-Bertani liquid medium can be used, or genetically engineered bacteria that can produce nanobodies after gene editing of Escherichia coli Nissle1917 according to the above method (1) can be used, corresponding to the OD of the bacterial liquid culture) are prepared. 600 (Greater than 0.2) were mixed at a volume ratio of 1:1 to form a core precursor solution. The core precursor solution was then injected into the hydrogel capsule shell prepared in Example 1 using a syringe. Since the hydrogel capsule shell had been soaked in CaCl2 solution, Ca... 2+ It can penetrate into the kernel, thereby cross-linking the kernel and forming Ca. 2+ The cross-linked sodium alginate hydrogel serves as the core, and the overall preparation process of the hydrogel is as follows: Figure 1 As shown, the probiotic delivery system based on the core-shell hydrogel structure was obtained.

[0062] Figure 4Cryo-electron microscopy images and pore area statistics of the probiotic delivery system based on core-shell hydrogel structure show that the pore area of ​​the hydrogel capsule shell is mostly concentrated below 0.1 square micrometers, with a pore size of 200–300 nm, which can isolate bacteria while allowing nutrients and therapeutic molecules secreted by bacteria to enter and exit.

[0063] Incorporating LPS selective removal agents into the outer shell hydrogel formulation further ensures the safety of the system when used in humans. Histidine was selected as the lipopolysaccharide (LPS) removal agent. Figure 6 Live bacteria were encapsulated in hydrogel capsule shells (containing different concentrations of histidine), and the LPS concentration leaked into the external solution was measured. It was found that the incorporation of histidine further reduced the LPS concentration in the external solution.

[0064] Figure 7 The therapeutic effect on tumors after subcutaneously implanting the above-mentioned probiotic delivery system based on core-shell hydrogel (the probiotic loaded is a genetically engineered bacterium that can produce nanobodies after gene editing of Escherichia coli Nissle1917 according to the above method (1)) into mice.

[0065] Specifically, an intestinal retention device used for the treatment of radiation colitis in pigs ( Figure 8 The dimensions are as follows:

[0066] Lid: Bottom: 10mm in diameter, 4mm thick;

[0067] Fitting part: 8mm in diameter, 6.6mm in inner diameter, 3*3mm opening;

[0068] Outer shell: 10mm in diameter, 8mm in inner diameter, 28mm in height, 3*3mm opening at the top, and 3.6*14mm opening in the middle (13mm from the bottom).

[0069] Assembled body: 10mm in diameter, 32mm in height;

[0070] The diameter of the rubber band thread inserted into the groove is 1-1.5mm;

[0071] This size can be adjusted according to the scenario where the device resides.

[0072] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A hydrogel capsule shell for probiotic delivery, characterized in that, The preparation method includes the following steps: A hydrogel precursor solution for the capsule shell was prepared using sodium alginate, acrylamide, a selective lipopolysaccharide remover, an initiator, and a first crosslinking agent. An accelerator was then added to induce the hydrogel to form a preliminary gel, thus preparing a capsule shell precursor. The capsule shell precursor was then subjected to multiple crosslinking processes in MES buffer containing a second crosslinking agent, an activator, and an activation enhancer, as well as in a solution containing calcium ions, to obtain the hydrogel capsule shell. The outer shell of the hydrogel capsule is a sealed shell, and the hollow part inside is used to load probiotics to isolate the probiotics from the outside world. The first crosslinking agent is selected from N,N'-methylenebisacrylamide or N,N'-bisacrylpiperazine; the second crosslinking agent is selected from adipate dihydrazide. The initiator is selected from ammonium persulfate or potassium persulfate; The accelerator mentioned is N,N,N',N'-tetramethylethylenediamine; The activator is selected as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; The activating and enhancing agent is selected from N-hydroxysuccinimide; Histidine is the selective remover of lipopolysaccharides. In the shell hydrogel precursor solution, the solvent is water, the mass fraction of sodium alginate is 2-3 wt%, the mass fraction of acrylamide is 20 wt%, the mass fraction of the first crosslinking agent is 0.05-0.08 wt%, the content of the initiator is 0.005-0.01 wt%, and the concentration of the lipopolysaccharide selective removal agent is 1-10 mM; the amount of accelerator added is 0.01-0.1 wt% of the shell hydrogel precursor solution.

2. The hydrogel capsule shell according to claim 1, characterized in that, In the shell hydrogel precursor solution, the mass fraction of sodium alginate is 2 wt% and the mass fraction of acrylamide is 20 wt%.

3. The hydrogel capsule shell according to claim 1, characterized in that, In the MES buffer containing a second crosslinking agent, an activator, and an activation enhancer, the concentration of the activator 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 5-8 mM, the concentration of the activation enhancer N-hydroxysuccinimide is 1.5-5 mM, and the concentration of the second crosslinking agent adipate dihydrazide is 5-15 mM.

4. The hydrogel capsule shell according to claim 1, characterized in that, The capsule shell precursor was placed in a MES buffer solution containing a second crosslinking agent, an activator, and an activation enhancer for crosslinking at 0-40°C for 0.5-1 h. After removal, it was placed in a solution containing calcium ions for crosslinking at 0-40°C for 0.5-1 h to obtain the hydrogel capsule shell.

5. The hydrogel capsule shell according to claim 1, characterized in that, The hydrogel capsule shell has an internal pore size of 200-300 nm, which can adsorb lipopolysaccharides produced by probiotics, allowing external nutrients to enter and probiotics to release therapeutic metabolites.

6. A probiotic delivery system based on a core-shell hydrogel, characterized in that, The structure includes the hydrogel capsule shell as described in claim 1 and a core containing probiotics; The method for preparing the probiotic-containing core includes: mixing sodium alginate solution and probiotic culture medium to obtain a core precursor solution; transferring the core precursor solution to the hollow part of the hydrogel capsule shell; allowing it to stand so that calcium ions in the hydrogel capsule shell can permeate into the core precursor solution and crosslink to form a core, thereby obtaining the probiotic delivery system based on the core-shell structure hydrogel. The probiotics mentioned are live bacteria that can secrete metabolic substances with therapeutic effects.

7. The probiotic delivery system based on a core-shell hydrogel according to claim 6, characterized in that, Sodium alginate solution and probiotic culture medium are mixed in equal volume ratio. The OD of the probiotic culture medium... 600 >0.2 indicates that the probiotic culture medium contains carbon sources, nitrogen sources, growth factors, inorganic salts, or osmotic pressure regulators that can be utilized by probiotics.

8. A drug-retention structure, characterized in that, Includes the probiotic delivery system and residence component based on the core-shell structured hydrogel as described in claim 6.

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