Probiotic delivery system based on core-shell structure hydrogel and application thereof

Abstract

The invention discloses a probiotic delivery system based on core-shell structure hydrogel and application of the probiotic delivery system, and belongs to the technical field of biological medicines.The probiotic delivery system based on the core-shell structure hydrogel comprises a hydrogel capsule shell and an inner core containing probiotics, the hydrogel capsule shell isolates the probiotics from the outside, and the inner core contains the probiotics; the hydrogel capsule comprises a shell and a core, the shell is made of sodium alginate, lipopolysaccharide generated by probiotics is adsorbed, the internal aperture of the shell of the hydrogel capsule is 200-300 nm, external nutrient substances are allowed to enter, metabolic substances secreted by the probiotics and having a treatment effect are allowed to go out, and the core is sodium alginate hydrogel which can well keep bacterial activity and contains certain nutrient substances required by bacteria. The probiotic delivery system has double isolation effects of physical isolation and chemical isolation, avoids negative effects of bacterial endotoxin on a human body, can realize bacterial isolation and drug permeation balance, and is good in long-term stability.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and particularly relates to a probiotic delivery system based on a core-shell structure hydrogel and applications thereof. Background Art

[0002] Drug reservoir systems, as a mature strategy for local drug delivery, play an important role in the management of chronic diseases, covering areas such as inflammatory diseases, endocrine disorders, and malignant tumors. Such systems generally include injectable controlled-release preparations and surgically implanted devices, which can achieve sustained release of therapeutic drugs, thereby reducing systemic exposure and improving target site specificity. Reduced dosing frequency helps maintain stable circulating drug concentrations and improve patient compliance. However, traditional drug reservoir systems have limitations in terms of total drug release. Once the drug load is exhausted, they lose their efficacy and cannot be refilled through non-invasive methods. These problems are more prominent when the implant site is difficult to access.

[0003] To address the dose limitations and refilling difficulties of drug reservoirs, the concept of living drug depots (LDDs) has emerged, which utilize living cells rather than pre-prepared drugs to achieve continuous production of therapeutic drugs. These cells can achieve customized and controllable drug delivery by secreting specific therapeutic molecules. Living cells encapsulated in LDDs can autonomously extract nutrients from the surrounding microenvironment, proliferate, and synthesize and secrete therapeutic molecules locally. This allows for continuous drug production without the need for external supplementation. Among the various cell types explored, bacteria have unique advantages due to their ease of culture, rapid proliferation rate, and relatively low nutrient requirements, making them particularly attractive candidates for living therapeutic modalities.

[0004] Despite the increasing use of live bacteria in therapeutic areas, the development of safe and stable drug reservoirs based on live bacteria still faces many challenges and has not made significant progress. This is because the safe and effective use of live bacteria in the human body requires both physical and chemical isolation. Physically, the bacteria must be restricted to prevent uncontrolled proliferation or premature elimination by the host immune system before the therapeutic effect is achieved; chemically, the endotoxins released during bacterial lysis must be neutralized, as these endotoxins may act as nonspecific immunogens and cause adverse symptoms such as fever; in addition, the implantable carrier used to carry the bacteria must have sufficient mechanical strength to prevent bacterial leakage and ensure safe and complete removal after the treatment is completed. This is crucial to reducing the risk of uncontrolled bacterial spread or disruption of the local microbial community.

[0005] Chinese patent publication number CN119223942A discloses a magnetic hydrogel-encapsulated bacterial biosensor platform. This invention uses a sodium alginate hydrogel to encapsulate a bacterial strain and magnetic Fe3O4 microparticles to form a biosensor for detecting intestinal inflammation. The bacterial strain reports the presence of the inflammatory marker hemoglobin through bioluminescent signal expression, and the presence of the magnetic Fe3O4 microparticles allows for efficient recovery of intact microspheres from feces by magnetic adsorption, providing a theoretical basis and new method for achieving efficient, rapid, and accurate in vitro detection and diagnosis of intestinal inflammation. Chinese patent publication number CN119632947A discloses a leak-free core-shell hydrogel microsphere delivery system for contactless microbiota transplantation. This invention uses a two-step photoinitiated emulsion polymerization of methacrylated gelatin to encapsulate bacteria in the core of the core-shell microspheres, constructing a contactless microbiota transplantation system that has demonstrated good therapeutic efficacy and safety.

[0006] However, the above-mentioned existing bacterial delivery systems have the following defects: (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 (generally 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 native flora in the body or cause infection due to uncontrolled proliferation or potential genetic mutations), and often limits the integration of additional functions; (2) A major pain point limiting the application of live bacteria in vivo is uncontrolled bacterial proliferation and spread. The toxins and certain metabolites produced by bacteria can induce infection. This is particularly fatal in the scenario of subcutaneous bacterial implantation, which directly hinders the development of subcutaneous implant 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 lipopolysaccharide can trigger systemic inflammation through TLR4-mediated cytokine storm. (3) The existing sodium alginate-acrylamide hydrogel has limited mechanical strength due to its swelling in a solution environment. If used in an in vivo environment, it will be difficult to maintain structural integrity and may cause bacteria to leak out and proliferate uncontrollably in the body, bringing a series of negative effects. Summary of the Invention

[0007] In order to address the deficiencies in the above-mentioned prior art, the present 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 can achieve a higher strength and more stable physical isolation effect, prevent the leakage of probiotics (bacteria), and prevent bacterial lipopolysaccharide from causing inflammation. The probiotic delivery system based on the core-shell hydrogel structure has broad application prospects in the LDD field.

[0008] The specific technical solutions adopted are as follows: A hydrogel capsule shell for probiotic delivery, the preparation method of which comprises the following steps: A shell hydrogel precursor solution is prepared using sodium alginate, acrylamide, a lipopolysaccharide selective remover, an initiator, and a first cross-linking agent, and an accelerating agent is added to initially gel the hydrogel to prepare a capsule shell precursor. The capsule shell precursor is sequentially placed in an MES buffer solution containing a second cross-linking agent, an activator, and an activation enhancer, and a solution containing calcium ions for multiple cross-linking to obtain the hydrogel capsule shell. The hydrogel capsule shell is a sealed shell, and the hollow portion inside is used to load the probiotics to isolate the probiotics from the outside world; The first cross-linking agent is selected from N,N'-methylenebisacrylamide or N,N'-bisacryloylpiperazine; the second cross-linking agent is selected from adipic acid dihydrazide; The initiator is ammonium persulfate or potassium persulfate; The speed increasing agent is N,N,N',N'-tetramethylethylenediamine; The activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; The activation enhancer is N-hydroxysuccinimide.

[0009] The shell of the hydrogel capsule for probiotic delivery provided by the present invention is a sodium alginate-polyacrylamide hydrogel incorporated with an LPS selective remover. The hydrogel has significantly improved mechanical properties and anti-swelling ability due to multiple cross-linking steps, and can achieve a higher strength and more stable physical isolation effect, preventing the leakage of probiotics (bacteria) and preventing inflammation caused by bacterial lipopolysaccharide.

[0010] 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).

[0011] 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 cross-linking 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 remover is 1-10 mM; the amount of the accelerator added is 0.01-0.1vt% of the shell hydrogel precursor solution.

[0012] After the accelerator is added, the shell hydrogel precursor solution will quickly solidify to form a capsule shell precursor.

[0013] Most preferably, the mass fraction of sodium alginate in the shell hydrogel precursor solution is 2 wt %, and the mass fraction of acrylamide is 20 wt %. Experiments have shown that the performance of the hydrogel capsule shell produced under the above parameters is optimal.

[0014] Preferably, in the MES buffer containing the second cross-linker, the activator and the 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 cross-linker adipic acid dihydrazide is 5-15 mM.

[0015] Preferably, the capsule shell precursor is placed in a MES buffer containing a second crosslinker, an activator and an activation enhancer for crosslinking at 0-40°C for 0.5-1 h, and then placed in a solution containing calcium ions for crosslinking at 0-40°C for 0.5-1 h to obtain the hydrogel capsule shell.

[0016] More preferably, the solution containing calcium ions is a CaCl2 solution with a concentration of 0.5-1.0 M.

[0017] Preferably, the inner pore size of the hydrogel capsule shell is 200-300 nm, which can absorb lipopolysaccharide produced by probiotics, allowing external nutrients to enter and metabolites with therapeutic effects secreted by probiotics to be delivered.

[0018] The present invention also provides a probiotic delivery system based on a core-shell hydrogel structure, which comprises the hydrogel capsule shell and a core containing probiotics; The preparation method of the core containing probiotics comprises: mixing a sodium alginate solution and a probiotic culture solution to obtain a core precursor solution, transferring the core precursor solution to the hollow portion of the hydrogel capsule shell, and allowing the calcium ions in the hydrogel capsule shell to penetrate into the core precursor solution and cross-link to form a core, thereby obtaining the probiotic delivery system based on the core-shell hydrogel structure; The probiotics are live bacteria (with or without genetic editing) that can secrete metabolites with therapeutic effects.

[0019] The aforementioned probiotic delivery system based on a core-shell hydrogel, as an enhanced LDD platform, uses an optimized delivery system (the present invention proposes a method for integrally encapsulating live bacteria in capsule- or rod-shaped core-shell hydrogels ("macroencapsulation"). This macroencapsulation technology facilitates complete post-treatment removal and has the potential to integrate auxiliary technologies such as drug delivery devices) to encapsulate microorganisms, adsorb bacterial lipopolysaccharides, and ensure continuous therapeutic secretion, minimizing immune responses. In animal models of tumors and radiation enteritis, both implantation and endoscopic administration of LDDs carrying different bacteria demonstrated sustained release, enhanced efficacy, and long-term safety. This delivery system provides a versatile and effective solution for long-term localized drug delivery, overcoming a key obstacle to live bacterial therapy.

[0020] Preferably, the sodium alginate solution and the probiotic culture solution are mixed in an equal volume ratio, and the OD of the probiotic culture solution is 600 >0.2, the probiotic culture medium contains carbon sources, nitrogen sources, growth factors, inorganic salts or osmotic pressure regulators that can be used by probiotics.

[0021] Further preferably, the probiotics are bifidobacteria, lactobacilli, lactococci, lactobacilli, lactobacilli, etc. (which can secrete short-chain fatty acids, bacteriocins and other substances that have the effect of inhibiting pathogens), or non-pathogenic Escherichia coli that have been genetically edited to secrete immune checkpoint inhibitors (PD-L1 nanoantibodies, CTLA-4 nanoantibodies, etc.), neutralizing antibodies to pro-inflammatory factors (NF-a and IL-23 bispecific antibodies, etc.).

[0022] Preferably, the core precursor solution is injected into the hollow portion of the hydrogel capsule shell obtained in step (1).

[0023] Preferably, the raw materials of the hydrogel capsule shell include acrylamide, sodium alginate and histidine. Polyacrylamide hydrogel is prepared using acrylamide as a monomer, ammonium persulfate as an initiator, and N,N'-methylenebisacrylamide as a cross-linking agent. Then, polyacrylamide and sodium alginate form a covalent bond in MES buffer containing a cross-linker and a catalyst, and are further soaked in 1.0 MCaCl2 to allow the sodium alginate to cross-link under the mediation of calcium ions to obtain the final hydrogel capsule shell.

[0024] Preferably, the core is a hydrogel precursor made by mixing sodium alginate and bacterial culture, and then 2+ The core is mediated to be cross-linked to form a sodium alginate hydrogel that can better maintain bacterial activity and contains certain nutrients required by bacteria.

[0025] Optionally, the probiotic delivery system based on the core-shell structure hydrogel is cylindrical, with a diameter of no more than 15 mm and a height of no more than 30 mm.

[0026] The present invention also provides a drug retention structure, comprising the probiotic delivery system based on the core-shell structure hydrogel and a retention component.

[0027] Furthermore, the drug retention structure can deliver probiotics to the skin, subcutaneous tissue, gastrointestinal tract, urogenital tract and other parts of the body for the treatment of tumors and various inflammatory diseases including radiation colitis.

[0028] Furthermore, the resident component is used to protect the probiotic delivery system based on the core-shell hydrogel structure and fix it to the intestinal lining.

[0029] Compared with the prior art, the present invention has the following beneficial effects: (1) The probiotic delivery system based on the core-shell hydrogel provided by the present invention has the dual isolation effects of physical isolation and chemical isolation: physical isolation of bacteria: the shell of the hydrogel capsule isolates the bacteria inside while allowing external nutrients and internal secretion products (such as PD-L1 nanoantibodies, TNF-a and IL-23 bispecific antibodies, GLP-1, etc.) to enter and exit, thereby achieving the effect of a local reservoir for continuous secretion of drugs; chemical isolation of LPS: histidine and the like 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 used in subcutaneous implantation scenarios).

[0030] (2) The core-shell hydrogel-based probiotic delivery system provided by the present invention is designed by combining a sodium alginate hydrogel core with a sodium alginate-polyacrylamide multi-crosslinked shell layer to achieve a balance between bacterial isolation and drug penetration. The sodium alginate-polyacrylamide outer layer has multiple crosslinking characteristics, including crosslinking between sodium alginate and polyacrylamide, crosslinking within polyacrylamide, and calcium ion-mediated crosslinking of sodium alginate, thereby achieving long-term stability of the system in scenarios such as simulated gastrointestinal tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a diagram of the preparation process of the probiotic delivery system based on core-shell structure hydrogel and actual photos of different sizes. The probiotic used is Escherichia coli Nissle1917.

[0032] Figure 2 This is a diagram showing the effects of different mass ratios of sodium alginate and acrylamide when preparing hydrogel capsule shells.

[0033] Figure 3 This is a comparison of the swelling properties of the hydrogel capsule shell before and after the calcium ion cross-linking step. SGF is a simulated gastric fluid (pH 1.5, containing enzyme), and SIF is a simulated intestinal fluid (pH 7.0, containing enzyme).

[0034] Figure 4 Cryo-electron microscopy images and pore area statistics of the probiotic delivery system based on core-shell structure hydrogel.

[0035] Figure 5 Comparison of the mechanical properties of the hydrogel capsule shell before and after the calcium ion cross-linking step (cyclic compression test).

[0036] Figure 6 This is a diagram showing the adsorption effect of the probiotic delivery system based on the core-shell hydrogel on bacterial LPS. Gel represents the group in which bacteria are loaded into the hydrogel capsule shell without histidine, and His-Gel represents the group in which bacteria are loaded into the hydrogel capsule shell containing different concentrations of histidine.

[0037] Figure 7 This is the therapeutic effect of the core-shell hydrogel-based probiotic delivery system on tumors after subcutaneous implantation in mice (Group 1 was not treated; Group 2 was implanted with a blank hydrogel capsule shell; Group 3 was injected with engineered bacteria through the tail vein; Group 4 was implanted with the core-shell hydrogel-based probiotic delivery system).

[0038] Figure 8 It is a real picture of the resident component and a real picture of the drug resident structure of the probiotic delivery system based on the core-shell structure hydrogel. DETAILED DESCRIPTION

[0039] The present invention will be further described below with reference to the following examples. It should be understood that these examples are intended only to illustrate the present invention and are not intended to limit the scope of the present invention.

[0040] Example 1 Preparation of hydrogel capsule shell for probiotic delivery (1) Prepare an aqueous solution containing 2 wt% sodium alginate and 20 wt% acrylamide. After fully dissolving, add histidine to make its final concentration in the shell hydrogel precursor solution 1 mM. Continue to add 0.01 wt% ammonium persulfate and 0.05 wt% N,N'-methylenebisacrylamide, mix thoroughly, remove bubbles, and obtain a shell hydrogel precursor solution. Pour the shell hydrogel precursor solution after removing bubbles into a mold, add 0.05% (vt / vt) of the gel volume accelerator N,N,N',N'-tetramethylethylenediamine (TEMED), wait for less than 15 minutes at room temperature to complete the initial gelation (acrylamide polymerization), and prepare a 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 crosslinker, an activator, and an activation enhancer, and crosslink at room temperature for 45 minutes. The MES buffer containing the second crosslinker, the activator, and the activation enhancer contains 8 mM activator 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), 5 mM activation enhancer N-hydroxysuccinimide (NHS), and 10 mM second cross-linker adipic acid dihydrazide (ADH) were added to form a covalent bond between sodium alginate and the polyacrylamide network. In order to further optimize the performance of the hydrogel capsule shell, the hydrogel capsule shell was immersed in a 1.0 MCaCl2 solution at room temperature for 45 minutes to obtain the hydrogel capsule shell for probiotic delivery.

[0041] When determining the hydrogel capsule shell formula, the ratio of sodium alginate and acrylamide was screened. We used 0-3 wt% sodium alginate and 5-10 wt% acrylamide, and finally determined that the hydrogel containing 2-3 wt% sodium alginate and 20 wt% acrylamide could better encapsulate bacteria ( Figure 2 ).

[0042] like Figure 3 As shown, the added cross-linking step of the present invention greatly reduces the swelling rate of the hydrogel capsule shell in the simulated gastrointestinal fluid (by about 50%), which is beneficial to the long-term stable application of the system in the gastrointestinal environment.

[0043] At the same time, the added calcium ion cross-linking step greatly increased the mechanical properties of the hydrogel capsule shell, as shown in the compression test results. Figure 5As shown. The sodium alginate-polyacrylamide hydrogel of the present invention can withstand a pressure of about 30 kPa when deformed by 60%. According to the pressure range in the gastrointestinal tract: esophagus: 4-8 kPa (peak value during swallowing); stomach: 0.5-2 kPa (basic peristalsis), 3-10 kPa (strong contraction or vomiting); small intestine: 1-5 kPa (segmental contraction and propulsive peristalsis); colon: 2-15 kPa (high-amplitude propagating contraction, such as during defecation). It can be concluded that the hydrogel capsule shell can fully withstand the pressure at various locations in the digestive tract, which is conducive to the application of the system in environments such as the gastrointestinal tract. In addition, the mechanical properties of the hydrogel system can also make it suitable for application scenarios such as implants and wearable devices.

[0044] Example 2 Probiotic delivery system based on core-shell hydrogel (1) Genetically edited bacteria can produce immune checkpoint inhibitors, such as PD-L1 nanoantibodies and CTLA-4 nanoantibodies (used in tumor treatment).

[0045] Plasmid design: After confirming that the nanobody binds to its respective target, the PD-L1 nanobody and CTLA-4 nanobody sequences are cloned into a high-copy plasmid for strong constitutive expression. tac The nanobody was cloned into a separate plasmid downstream of the promoter to achieve maximum gene expression. 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 the vector to prevent plasmid loss during bacterial replication. For details, please refer to the reference 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 Preparation of genetically engineered bacteria secreting PD-L1 nanoantibodies and CTLA-4 nanoantibodies.

[0046] (2) Genetically edited bacteria can produce neutralizing antibodies against pro-inflammatory factors, such as tumor necrosis factor-α (TNF-α) neutralizing nanoantibodies (targeting inflammation).

[0047] Editing method: Non-pathogenic Escherichia coli (EcN or HS strain) was modified by synthetic biology: First, the pathogenic ipa operon of the Shigella type III secretion system (T3SS) was deleted, and only the mxi-spa structural gene cluster was retained and integrated into a specific chromosomal site, so that the T3SS was converted from a host cell injection mode to an environmental secretion mode; secondly, the constitutive promoter PJ23119 was used to drive the expression of the transcriptional regulatory factor VirB to achieve continuous activation of the secretion system; then, the OspC2 secretion signal peptide was fused to the N-terminus of the therapeutic nanoantibody (such as anti-TNF-α) to ensure efficient secretion of the heterologous protein and maintain functional activity; finally, the host alr / dadX gene was knocked out, and the alr compensation gene was introduced into the expression vector to construct a stable symbiotic system without antibiotic screening, so as to achieve long-term colonization of the engineered bacteria in the intestine and continuous secretion of therapeutic proteins. For details, please refer to the literature Jason P. Lynch, Coral González-Prieto, Analise Z. Reeves, et al . Engineered Escherichia coli for the in situ secretion of therapeutic nanobodies in the gut. Cell Host & Microbe 31, 634-649 (2023). https: / / doi.org / 10.1016 / j.chom.2023.03.007. Preparation of genetically engineered bacteria secreting tumor necrosis factor-α (TNF-α) neutralizing nanobodies.

[0048] The preparation process of the hydrogel core is as follows: first, 5 wt% sodium alginate solution and bacterial culture medium (specifically, Escherichia coli Nissle1917 in Luria-Bertani liquid culture 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)) are prepared, and the OD of the corresponding bacterial liquid culture is 0. 600 greater than 0.2) were mixed in a volume ratio of 1:1 to form a core precursor solution, and then the core precursor solution was injected into the hydrogel capsule shell prepared in Example 1 through a syringe. Since the hydrogel capsule shell was soaked in CaCl2 solution, Ca 2+ It will penetrate into the core and cross-link the core to form Ca 2+ The cross-linked sodium alginate hydrogel is used as the core. The overall preparation process of the hydrogel is as follows: Figure 1 As shown, the probiotic delivery system based on the core-shell structure hydrogel was obtained.

[0049] Figure 4The cryo-electron microscopy images and pore area statistics of the probiotic delivery system based on core-shell hydrogels show that the pore area of ​​the hydrogel capsule shell is mostly concentrated below 0.1 square microns, 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.

[0050] Incorporating LPS selective removers into the shell hydrogel formulation can further ensure the safety of the system when applied to the human body. Histidine was selected as the lipopolysaccharide (LPS) remover. Figure 6 Live bacteria were encapsulated in a hydrogel capsule shell (containing different concentrations of histidine) and the LPS concentration leaked into the external solution was measured. It was found that the LPS concentration in the external solution was further reduced after the incorporation of histidine.

[0051] Figure 7 The therapeutic effect on tumors was evaluated after the above-mentioned probiotic delivery system based on the core-shell hydrogel was implanted subcutaneously in mice (the loaded probiotics were genetically engineered bacteria capable of producing nanoantibodies after gene editing of Escherichia coli Nissle1917 according to the above method (1)).

[0052] Specifically, an intestinal resident device ( Figure 8 ) dimensions are as follows: Lid: Lid bottom: diameter 10mm, thickness 4mm; Fitting: diameter 8mm, inner diameter 6.6mm, opening 3*3mm; Housing: 10mm diameter, 8mm inner diameter, 28mm high, top opening 3*3mm, middle opening (13mm from the bottom, 3.6*14mm); Assembly: diameter 10mm, height 32mm; The diameter of the rubber band inserted into the groove is 1-1.5mm; This size can be adjusted based on the scenario in which the device resides.

[0053] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A hydrogel capsule shell for probiotic delivery, characterized in that The preparation method comprises the following steps: A shell hydrogel precursor solution is prepared using sodium alginate, acrylamide, a lipopolysaccharide selective remover, an initiator, and a first cross-linking agent, and an accelerating agent is added to initially gel the hydrogel to prepare a capsule shell precursor. The capsule shell precursor is sequentially placed in an MES buffer solution containing a second cross-linking agent, an activator, and an activation enhancer, and a solution containing calcium ions for multiple cross-linking to obtain the hydrogel capsule shell. The hydrogel capsule shell is a sealed shell, and the hollow portion inside is used to load the probiotics to isolate the probiotics from the outside world; The first cross-linking agent is selected from N,N'-methylenebisacrylamide or N,N'-bisacryloylpiperazine; the second cross-linking agent is selected from adipic acid dihydrazide; The initiator is ammonium persulfate or potassium persulfate; The speed increasing agent is N,N,N',N'-tetramethylethylenediamine; The activator is 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; The activation enhancer is N-hydroxysuccinimide.

2. The hydrogel capsule shell according to claim 1, characterized in that The lipopolysaccharide selective remover is an amino acid or a polycationic substance. The amino acid includes histidine, lysine or arginine, and the polycationic substance includes polyethyleneimine, poly-L-lysine or poly-L-histidine.

3. The hydrogel capsule shell according to claim 1, wherein 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 cross-linker is 0.05-0.08 wt%, the content of the initiator is 0.005-0.01 wt%, and the concentration of the lipopolysaccharide selective remover is 1-10 mM; the amount of the accelerator added is 0.01-0.1vt% of the shell hydrogel precursor solution.

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

5. The hydrogel capsule shell according to claim 1, wherein In the MES buffer containing the second cross-linker, activator, and 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 cross-linker adipic acid dihydrazide is 5-15 mM.

6. The hydrogel capsule shell according to claim 1, wherein The capsule shell precursor is placed in a MES buffer containing a second crosslinker, an activator and an activation enhancer for crosslinking at 0-40°C for 0.5-1 h, and then placed in a solution containing calcium ions for crosslinking at 0-40°C for 0.5-1 h to obtain the hydrogel capsule shell.

7. The hydrogel capsule shell according to claim 1, wherein The inner pore size of the hydrogel capsule shell is 200-300 nm, which can absorb lipopolysaccharide produced by probiotics, allowing external nutrients to enter and metabolites with therapeutic effects secreted by probiotics to be delivered.

8. A probiotic delivery system based on a core-shell hydrogel, characterized in that: The structure comprises the hydrogel capsule shell of claim 1 and a core containing probiotics; The preparation method of the core containing probiotics comprises: mixing a sodium alginate solution and a probiotic culture solution to obtain a core precursor solution, transferring the core precursor solution to the hollow portion of the hydrogel capsule shell, and allowing the calcium ions in the hydrogel capsule shell to penetrate into the core precursor solution and cross-link to form a core, thereby obtaining the probiotic delivery system based on the core-shell hydrogel structure; The probiotics are live bacteria that can secrete metabolites with therapeutic effects.

9. The probiotic delivery system based on core-shell structure hydrogel according to claim 8, characterized in that: Sodium alginate solution and probiotic culture solution were mixed in equal volume ratio, and the OD 600 >0.2, the probiotic culture medium contains carbon sources, nitrogen sources, growth factors, inorganic salts or osmotic pressure regulators that can be used by probiotics.

10. A drug retention structure, characterized in that: The probiotic delivery system comprises the core-shell structure hydrogel-based probiotic delivery system according to claim 8 and a resident component.

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