Intestinal targeting pH-ROS double-response hydrogel-LGG-coated EGCG-Mn packaging system and preparation and application thereof

Abstract

The invention firstly provides an intestinal tract targeted pH-ROS double-response hydrogel packaging system, the hydrogel packaging system is sodium alginate (SA)-based hydrogel modified by ketothioacetal (tK) and calcium ions, and the hydrogel is used for intestinal tract probiotics lactobacillus rhamnosus GG (LGG). Wherein the hydrogel in unit volume contains 10 < 8 >-10 < 9 > CFU of LGG (Ligusticum Glycol Glycol); the LGG is the LGG modified by EGCG-Mn nano particles, namely LGG (at) EGCG-Mn. The hydrogel matrix adopted in the packaging system not only provides enough gastric acid resistance for the LGG, but also promotes colon targeting and enhances intestinal retention. More importantly, the modification of the EGCG-Mn nanoparticles endows the LGG with a cell protection capability in an excessive ROS environment, so that targeted release, stable survival and colonization of the LGG after the LGG reaches the colon are realized. The technical problems that the LGG cannot reach the colon sufficiently and is difficult to survive stably in the prior art are solved, and an unexpected technical effect is generated; and the method is simple to operate, quick in reaction and mild in reaction condition, and has important practical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of microbial materials and relates to encapsulation and application of probiotics, and specifically to a pH-ROS dual-responsive hydrogel encapsulation system for Lactobacillus rhamnosus and a preparation method and application thereof. Background Art

[0002] Ulcerative colitis (UC) is an inflammatory bowel disease (IBD) characterized by inflammation of the colonic mucosa, which extends to varying degrees from the rectum to the colon. The disease has a relapsing and remitting process, producing symptoms such as abdominal pain, diarrhea, and rectal bleeding. The global prevalence of UC continues to rise, and is expected to increase from 1.7 million cases in 2019 to 2 million cases in 2029, with an annual growth rate of 1.36%; at the same time, the annual cure rate of UC is disappointingly low. Increasing evidence confirms that the pathogenesis of UC is closely related to oxidative damage, abnormal immune response, intestinal mucosal barrier damage, and intestinal microbiota imbalance. Therefore, strategies such as dietary adjustments, probiotic and prebiotic supplements, and traditional drug treatments such as 5-aminosalicylic acid, corticosteroids, immunomodulators, and biologics are currently used to control colitis.

[0003] Oral probiotic therapy has gained great appeal as a means of treating UC in terms of safety, gut health, and immune support. Compared with traditional treatments such as corticosteroids and immunomodulators, probiotics are believed to have fewer side effects. This is because probiotics are believed to support the immune system by increasing the activity of macrophages or natural killer cells, rather than suppressing it, thereby minimizing the risk of infection. In addition, they can produce antimicrobial compounds, including lactic acid, acetic acid, hydrogen peroxide, and bacteriocins, which help restore the balance of the intestinal microbiota. As a commercial probiotic strain, Lactobacillus rhamnosus GG is able to enhance intestinal barrier function and modulate immune responses, making it a preferred probiotic for the treatment of colitis. It has been shown that LGG can effectively reduce inflammation and promote intestinal health in patients with colitis. However, due to the survival rate of LGG in the gastrointestinal tract (GIT) and its compliance with the intestinal mucosa, oral administration of LGG cannot be effectively achieved at present. The primary problem is that gastric acid with a pH of 2 significantly reduces the activity of LGG, thereby affecting the therapeutic effect.

[0004] To overcome this problem, researchers have proposed bulk encapsulation methods and single-cell encapsulation methods to ensure that a sufficient number of live probiotics are delivered to the target site. 2 +), a hydrogel is formed that exhibits remarkable pH-responsive behavior. At pH 2-3, the carboxyl groups in the alginate chains are protonated, resulting in reduced electrostatic repulsion and causing the hydrogel to shrink. In contrast, at pH 5-9, these carboxyl groups are deprotonated, increasing electrostatic repulsion and causing the hydrogel to swell as it absorbs more water. This pH-responsive property is expected to be used to encapsulate LGG, protecting them in the acidic gastric environment and releasing them in the intestine (pH 6-8). However, SA-Ca in the small intestine (pH 6.8) 2+ Premature release of hydrogel-encapsulated LGG may result in insufficient amount of LGG reaching the colon, thus affecting the overall efficacy of the treatment.

[0005] Moreover, one of the pathological characteristics of UC is the imbalance of gastrointestinal inflammatory homeostasis, which will lead to the generation of a large amount of reactive oxygen species (ROS) in the lesions, far exceeding the normal range; thus damaging the cell membrane and DNA of LGG, reducing the survival and intestinal colonization ability of LGG. How to make a sufficient amount of LGG reach the colon and release it, and how to stably survive in the colon, is a technical problem that needs to be solved in the existing technology of using LGG to treat UC, and there is no relevant report yet. Summary of the invention

[0006] In view of the problems existing in the oral administration of LGG for the treatment of UC in the prior art, the present invention provides a pH-ROS dual-responsive hydrogel LGG encapsulation system. The hydrogel matrix used in the encapsulation system not only provides LGG with sufficient gastric acid resistance, but also promotes colon targeting and enhances intestinal retention. More importantly, the LGG encapsulated in the above-mentioned hydrogel matrix is ​​LGG modified with EGCG-Mn nanoparticles (LGG@EGCG-Mn), which gives LGG the ability to protect cells in an excessive ROS environment, thereby achieving targeted release, stable survival, and colonization of LGG after reaching the colon.

[0007] The technical solution of the present invention:

[0008] Intestinal targeted pH-ROS dual-responsive hydrogel encapsulation system, the hydrogel encapsulation system is a sodium alginate (SA)-based hydrogel modified with ketothiol (tK) and calcium ions, and the hydrogel is used for intestinal probiotics; the intestinal probiotics are Lactobacillus rhamnosus GG (LGG). Through the modification of calcium ions, the hydrogel has pH response. In addition, tK releases free thiols through ROS-triggered degradation, and the binding affinity of LGG to the intestinal lining is improved through the interaction of the thiol mucosal surface. Therefore, the pH-ROS dual-responsive hydrogel encapsulation system not only provides sufficient gastric acid resistance for probiotics, but also promotes colon targeting and enhances intestinal retention.

[0009] The pH-ROS dual-responsive hydrogel encapsulation system as described above was prepared by the following method: (1) SA, EDC and NHS were added to water and stirred to activate the carboxyl groups on the SA molecules to obtain solution I; wherein the molar ratio of SA, EDC and NHS was 2:1:1 to 2:2:1. (2) An appropriate amount of tK was added to solution I and stirred until the system showed slight cross-linking, thereby obtaining a SA-tK semifluid; wherein the molar ratio of SA to tK was 50:1 to 100:1. (3) The SA-tK semifluid was mixed with an appropriate amount of 10 to 20 mg / mL CaCl2 solution and stirred for 30 seconds to obtain a SA-tK-CaCl2 solution. 2+ Hydrogel. Wherein, the volume of SA-tK semifluid is not greater than the volume of CaCl2 solution.

[0010] A Lactobacillus rhamnosus (LGG) hydrogel encapsulated preparation for the treatment of ulcerative colitis, wherein the LGG hydrogel encapsulated preparation comprises LGG and a hydrogel encapsulating LGG; the hydrogel is the pH-ROS dual-responsive hydrogel as described above. The hydrogel contains 10 8 -10 9 CFU of LGG; the LGG is LGG modified with EGCG-Mn nanoparticles, i.e., LGG@EGCG-Mn; wherein the EGCG-Mn is not less than three times the amount of LGG. Epigallocatechin gallate (EGCG is a bioactive polyphenol with a recognized therapeutic effect on a variety of inflammations, but due to its instability, it usually needs to be modified to maintain its pharmacological efficacy. In the present application, the EGCG is modified with Mn via a trihydroxyphenyl (gallic alcohol) group. 2+ The nanoparticles formed pentagonal ring coordination bonds and then self-aggregated EGCG-Mn nanoparticles on the surface of LGG, thus serving as an antioxidant barrier to protect LGG from oxidative stress.

[0011] The preparation method of the LGG hydrogel encapsulation preparation as described above includes the following steps: (1) preparing LGG modified with EGCG-Mn nanoparticles, i.e., LGG@EGCG-Mn, by in situ deposition; through the modification of EGCG-Mn nanoparticles, not only the scavenging ability of LGG for ROS is significantly enhanced, but also the cell protection ability of LGG in an excessive ROS environment is given. (2) LGG@EGCG-Mn is mixed and stirred with SA-tK semi-fluid to obtain LGG@EGCG-Mn@SA-tK in a semi-fluid state. (3) Finally, an appropriate amount of CaCl2 solution is added and stirred for 30 seconds to obtain hydrogel-encapsulated LGG@EGCG-Mn, i.e., LGG@EGCG-Mn@SA-tK-CaCl2. 2+ , i.e., LGG hydrogel encapsulation preparation.

[0012] Wherein, the step (1) is specifically as follows: (a) incubating LGG in MRS culture medium at 37°C for 12 hours to obtain a LGG suspension; (b) taking a volume of V of the LGG suspension, centrifuging at 4-20°C and 5000rpm for 5 minutes. Removing the supernatant to obtain a cell precipitate, i.e., LGG; (c) sequentially adding a MnCl2·4H2O solution and an EGCG solution with a volume of V / 2 to LGG, shaking at 37°C and 600-1000rpm / min for 5 minutes, centrifuging at 4-20°C and 5000rpm for 5 minutes, removing the supernatant to obtain a precipitate, i.e., obtaining LGG@EGCG-Mn; wherein the concentration of the MnCl2·4H2O solution is 0.055-0.22mg / mL, and the concentration of the EGCG solution is 0.125-0.50mg / mL. In the present application, after the MnCl2·4H2O solution and the EGCG solution are added in sequence, LGG@EGCG-Mn can be obtained by a "one-pot" reaction. It is not only simple to operate and quick to react, but also has mild reaction conditions and has great application prospects.

[0013] Preferably, the precipitate is washed with HEPES in both step (b) and step (c), wherein step (b) is for removing excess culture fluid, and step (c) is for removing excess reactants.

[0014] The use of the Lactobacillus rhamnosus (LGG) hydrogel encapsulated preparation as described above in the treatment of ulcerative colitis.

[0015] Beneficial effects of the present invention:

[0016] (1) The present application first provides an intestinal-targeted pH-ROS dual-responsive hydrogel, which can provide probiotics with gastric acid resistance, smoothly reach the colon, achieve colon targeting, and enhance the survival ability of probiotics in the colon, with broad application prospects.

[0017] (2) In the present application, the pH-ROS dual-responsive hydrogel is wrapped around LGG modified with EGCG-Mn nanoparticles to obtain a Lactobacillus rhamnosus (LGG) hydrogel encapsulated preparation; this not only significantly enhances the ROS scavenging ability of LGG, but also gives LGG the ability to protect cells in an environment with excessive ROS.

[0018] (3) The present application provides a method for preparing the hydrogel encapsulation preparation, and the "one-pot method" is used to obtain LGG@EGCG-Mn. It is not only simple to operate and quick to react, but also has mild reaction conditions and has great application prospects.

[0019] (4) The Lactobacillus rhamnosus (LGG) hydrogel encapsulated preparation described in the present application overcomes the technical problems in the prior art that LGG cannot reach the colon in sufficient quantities and is difficult to survive stably, and is expected to be used for the treatment of ulcerative colitis, which has important social significance and huge economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Attached Figure 1 Preparation and screening of antioxidant coatings for LGG. A) Demonstration of the synthesis process of LGG@EGCG-Mn by probiotic-assisted EGCG-Mn nanoparticle deposition strategy; B) Transmission electron microscopy (TEM) images of LGG / EGCG and LGG@PDA; C) LGG on EGCG (without adding Mn 2+ ) Survival and growth after coating.

[0021] Attached Figure 2 Synthesis and characterization of LGG@EGCG-Mn. A) The size of LGG and LGG@EGCG-Mn measured by DLS, the error bars represent the standard error of the mean (n=3); B) Inverted fluorescence microscopy image of LGG@EGCG-Mn; C) 3D CLSM image of LGG@EGCG-Mn, EGCG-Mn was labeled with rhodamine B, scale bar: 50 μm; D) TEM images of LGG and LGG@EGCG-Mn, scale bar: 1 μm; E) HAADF-STEM image of LGG@EGCG-Mn, scale bar: 1 μm; F) Bacterial plates counted for LGG and LGG@EGCG-Mn; G) Statistical analysis of bacterial viability; H) Growth curves of LGG and LGG@EGCG-Mn; I) Flow cytometry analysis of LGG and LGG@EGCG-Mn.

[0022] Attached Figure 3Characterization of the in vitro ROS scavenging and cell protection capabilities of LGG@EGCG-Mn. A) DPPH· scavenging activity displayed by LGG, LGG@EGCG-Mn and LGG@PDA (left: UV-VIS spectral analysis, right: DPPH· elimination rate); B) LGG and LGG@EGCG-Mn exhibited DPPH· scavenging activity at different concentrations of EGCG-Mn (left: UV-VIS spectral analysis, right: DPPH· elimination rate); C) The total antioxidant capacity of LGG, LGG@EGCG-Mn and LGG@PDA was measured by the ABTS method (T-AOC assay kit), using trolox as a standard (Left: UV-Vis spectrophotometry analysis, right: Trolox equivalent antioxidant capacity); D) The total antioxidant capacity of LGG coated with different concentrations of EGCG-Mn was measured by ABTS method (T-AOC assay kit) with trolox as the standard (left: UV-Vis spectrophotometry analysis, right: Trolox equivalent antioxidant capacity); E) The total antioxidant capacity of LGG, LGG@EGCG-Mn and LGG@PDA was measured by FRAP method (T-AOC assay kit) with FeSO4·7H2O as the standard and 0.15mM trolox was used as a control (left: UV-visible spectrophotometer spectral analysis, right: FeSO4·7H2O concentration); F) The total antioxidant capacity of LGG coated with different concentrations of EGCG-Mn was measured by the FRAP method (T-AOC assay kit), with FeSO4·7H2O as the standard and 0.15mM trolox as the control (left: UV-VIS spectral analysis; right: FeSO4·7H2O concentration); G) Effects of EGCG and DA on the growth of different bacteria (LGG: Lactobacillus rhamnosus GG, EC: Escherichia coli; SA: Staphylococcus aureus); H) Cell viability of LGG and LGG@EGCG-Mn under different H2O2 concentrations (1, 0.5, 0.1mM); I) Growth curves of uncoated LGG and LGG@EGCG-Mn at different EGCG addition concentrations after incubation with 1mM H2O2 for 2 hours; J, K) EGCG-Mn and LGG@EGCG-Mn coated with 1mM H2O2 for 2 hours. Photos and counts of bacterial colonies formed on MRS agar plates of uncoated LGG and LGG@EGCG-Mn with different EGCG concentrations after incubation with H2O2 for 2 hours; uncoated LGG was used in PBS as a control, and the dilution factor of the bacterial solution was 10 6 Data are expressed as mean ± SD (n = 3 independent experiments), and significance was assessed using Student's t test and one-way analysis of variance (ANOVA) with Tukey's post hoc test to obtain p values, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

[0023] Attached Figure 4 SA-tK@Ca dual-responsive to ROS and pH 2+ Preparation of hydrogel. A) Schematic diagram of the process of synthesizing SA-tK semifluid; B) FT-IR results of SA-tK and SA; C) SA-tK semifluid and SA-tK-Ca 2+ Hydrogel, SA solution and SA-Ca 2+ D) SA-tK-CaCl2 solution formed by SA-tK and different concentrations of CaCl2 solution in a volume ratio of 1:1. 2+ Rheological properties of hydrogels, SA-Ca 2+ Hydrogel and SA-tK semifluid as controls; E) SA-tK-Ca 2+ Hydrogel (top) and SA-Ca 2+ SEM of hydrogel (bottom); F) Corresponding size distribution analysis fitted in Origin 2018; G) Effect of CaCl2 concentration and volume ratio (SA-tK / CaCl2) on gelation time; H) SA-tK-Ca 2+ Hydrogel and SA-Ca 2+ Photographs of hydrogel decomposition in a pH / ROS environment.

[0024] Attached Figure 5 The drug resistance and response of LGG with different encapsulation systems in simulated gastric acid SGF in vitro. Among them: A) LGG@EGCG-Mn@SA-tK-Ca 2+ CLSM images; the green channel shows LGG labeled with FITC, and the red channel shows EGCG-Mn labeled with rhodamine B; B) After incubation with pepsin-supplemented SGF (pH 2) for 1 h, LGG, LGG@EGCG-Mn, and LGG@EGCG-Mn@SA-tK-Ca 2+ Photo of bacterial colonies formed on MRS agar plates; LGG in PBS was used as a control, and the dilution factor of the bacterial solution was 0.5×10 6 ; C, D, E) After incubation with SGF containing pepsin (pH 2) at 37 °C and 200 rpm for a predetermined time (0.5, 1, 2 h), the activity of LGG, LGG@EGCG-Mn, and LGG@EGCG-Mn@SA-tK-Ca was measured by plate counting 2+ CFU is expressed as colony forming unit, and the data are presented as mean ± SD (n = 3). Student's t test was used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001; F) After incubation with pepsin-supplemented SGF (pH 2) for 1 h, the survival rate of LGG, LGG@EGCG-Mn and LGG@EGCG-Mn@SA-tK-Ca2+ Flow cytometry analysis was performed; LGG in PBS was used as a control; G) LGG, LGG@EGCG-Mn, and LGG@EGCG-Mn@SA-tK-Ca were stained with DAPI and PI before and after treatment in SGF (pH 2) containing pepsin for 1 hour. 2+ Representative fluorescence images of SA-tK-Ca under different conditions; H, I) 2+ Hydrogel and SA-Ca 2+ Release kinetics of probiotics from hydrogels.

[0025] Attached Figure 6 Enhanced adhesion of coated probiotics in vivo. A) Schematic diagram of experimental schedule; B) Oral administration of EGCG-Mn nanoparticles, LGG@EGCG-Mn and LGG@EGCG-Mn@SA-tK-Ca to healthy mice 2+ IVIS images 1, 5, 9, and 24 hours after surgery; C) IVIS detection of EGCG-Mn nanoparticles, LGG@EGCG-Mn, and LGG@EGCG-Mn@SA-tK-Ca retained in the GIT of healthy mice 2+ In vitro fluorescence images at 24 h, n = 3; D) Schematic diagram of the experimental timeline; E) LGG@EGCG-Mn@SA-tK-Ca 2+ Fluorescence images of the isolated intestine of mice treated with UC at 4 and 8 hours; 2+ Fluorescent images of mouse colonic circular sections at 8 hours post-injection; white represents Cy5.5-labeled LGG, scale bar 500 μm.

[0026] Attached Figure 7 GG@EGCG-Mn@SA-tK-Ca 2+Preventive effect in DSS-induced colitis model mice. A) Schematic diagram of experimental operation; B) Photos of colons harvested from mice in different groups on day 14; C) Changes in body weight of mice in different groups over time; D) Changes in DAI of mice in different groups over time; E) Spleen index of mice after different treatments (data collected immediately after mice were sacrificed); F) Quantitative length of colons harvested from mice in different groups on day 14; G) Representative images of H&E staining of colon tissues harvested from different groups on day 14, scale bar: 100 μm; H) Expression of mRNA encoding proinflammatory cytokines (IL-1β, TNF-α, and IL-6); I) Average optical density of immunofluorescence staining. Data are presented as mean ± SD (n = 5 for panels B to F, n = 3 for panels H and I), and statistical analysis was performed using one-way analysis of variance (ANOVA) and Student's t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. ns represents not significant. J) Representative immunofluorescence staining images. Scale bar: 200 μm

[0027] Attached Figure 8 Figure 1: Gut microbiome analysis by 16S rRNA sequencing and analysis of the levels of 7 different SCFAs in the intestine. A, B) OUT richness and Shannon α diversity index; C, D) PCoA and NMDS for β diversity determination; E) Representative bacterial taxonomic analysis at the level of intestinal microbial phyla; F) Heat map of the relative abundance of the top 30 families in the intestinal microbiota; G) LEfSe histogram, bacterial taxa that met the LDA score criteria > 2 were considered as biomarker taxa; H) Composition of the mouse intestinal flora at the genus level; I) Relative abundance of Clostridia_UCG-014, Akkermansia-muciniphils, and Lachnospiraceae. J) Analysis of SCFAs content in the colon. Data are presented as mean ± SD (n = 3 for the model group, n = 5 for the other five groups in Figures A to I, and n = 3 for Figure J). Statistical analysis was performed using one-way analysis of variance (ANOVA) and Student's t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. ns means not significant. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with the embodiments.

[0029] Raw materials: All were purchased from commercial sources.

[0030] Among them, epigallocatechin gallate (EGCG), sodium alginate (SA, 3-5, 15-60, 1000-1200, 7000-10000 mpa·s), 1-ethyl-(3-dimethylaminopropyl) semicarbazide hydrochloride (EDC), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) were all from Macklin. Cy5.5-NHS and 2-[2-(2-aminoethylthio)propane-2-ylthio]ethylamine (tK-NH2) were both from Chongqing Yusi Pharmaceutical Co., Ltd. Manganese chloride tetrahydrate (MnCl2·4H2O) and pepsin powder were both from Aladdin. Calcium chloride (CaCl2) was from Sinopharm Chemical Reagent Co., Ltd.

[0031] Dopamine (DA) was from Sigma-Aldrich. TRIS·HCl and agar were from MDbio. Dextran sulfate sodium salt (DSS, molecular weight 36,000 to 50,000 Daltons) was from Meilun Biotechnology. Fluorescein isothiocyanate (FITC), N-hydroxysuccinimide (NHS), 4′,6-diamino-2-phenylindole (DAPI), propidium iodide (PI), rhodamine B, DMEM cell culture medium, trypsin-EDTA solution, and phosphate buffered saline (PBS) were all purchased from Solarbio. MRS medium was from Hopebio. 1,1-Diphenyl-2-nitrohydrazine (DPPH) was from TCI-SCT.

[0032] Total antioxidant capacity assay kits (ABTS, FRAP), cytokine (IL-6, TNF-α and IL-10) detection ELISA kits, and CCK-8 solution were all from Bio-Tech. Fetal bovine serum (FBS) was from Wuhan Pronosai Biotechnology Co., Ltd. A magnetic bead-based cell tissue total RNA extraction kit was purchased from Biotech. Anti-ZO-1 tight junction protein antibody and anti-Occludin antibody were from Wuhan Boster Biotechnology Co., Ltd. HiScript III first-strand cDNA synthesis kit (+gDNA cleanup agent) and ChamQ Universal SYBR qPCR master mix were both from Vazyme.

[0033] Strains and culture conditions. The strain lactic acid bacteria (Lactobacillus rhamnosus GG, LGG, ATCC 53103) was purchased from the China Industrial Microbiological Culture Collection. The freeze-dried strain was revived overnight after adding 5 ml of culture medium. Then 1% LGG was inoculated into 10 ml of MRS liquid culture medium and left to stand at 37°C overnight.

[0034] Cells. Mouse macrophages (RAW264.7) and mouse lung fibroblasts (L929) were cultured in DMEM medium supplemented with 10% FBS at 37°C in a 5% CO2 atmosphere.

[0035] Animals. SPF male C57BL / 6N mice (6–8 weeks old, 20 ± 2 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. All animal experiments were conducted under the guidance and approved by the Animal Ethics Committee of Ocean University of China and Qingdao Municipal Hospital.

[0036] Example 1: Preparation of LGG@EGCG-Mn.

[0037] LGG was incubated in commercial MRS culture medium at 37°C for 12 hours to obtain LGG suspension (~10 9 CFU / mL)). Then, 1 mL of LGG suspension was centrifuged at 4°C and 5000 rpm for 5 minutes, and the supernatant was removed to obtain a cell precipitate, namely LGG. The cell precipitate was washed with HEPES; then 0.5 mL of MnCl2·4H2O solution (0.055 mg / mL) and 0.5 ml of EGCG solution (0.125 mg / mL) were added in sequence, and the mixture was vigorously shaken at 37°C and 900 rpm / min for 5 minutes. Centrifuged at 10°C and 5000 rpm for 5 minutes, and the supernatant was removed to obtain a precipitate. The precipitate was then washed with HEPES to remove excess reactants to obtain LGG@EGCG-Mn. Figure 1 As shown in A.

[0038] At the same time, LGG / EGCG and LGG@PDA were prepared as control samples. Specifically, 0.5 ml of the above-obtained LGG suspension and 0.5 ml of a single phenol solution (EGCG or DA) with a concentration of 0.125 mg / mL were mixed in Tris-HCl buffer (10 mM, pH 8.5), and oscillated in a dark environment for 0.5 h. Centrifuge at 4°C and 5000 rpm for 5 minutes, remove the supernatant to obtain a precipitate. The precipitate was then washed twice with PBS to remove excess reactants. The obtained LGG / EGCG and LGG@PDA were characterized by transmission electron microscopy (Scios, USA), as shown Figure 1 As shown in B.

[0039] Example 2: Preparation of LGG@EGCG-Mn.

[0040] Different from Example 1,

[0041] 1mL LGG suspension was centrifuged at 10℃ and 5000rpm for 5 minutes, and the supernatant was removed to obtain a cell precipitate, namely LGG. Then 0.5mL MnCl2·4H2O solution (0.11mg / mL) and 0.5ml EGCG solution (0.25mg / mL) were added in sequence, and the mixture was vigorously shaken at 600rpm / min for 5min at 37℃. Centrifuged at 20℃ and 5000rpm for 5 minutes, and the supernatant was removed to obtain a precipitate. The precipitate was washed with HEPES to remove excess reactants to obtain LGG@EGCG-Mn.

[0042] Example 3: Preparation of LGG@EGCG-Mn.

[0043] Different from Example 1,

[0044] 1mL LGG suspension was centrifuged at 20℃ and 5000rpm for 5 minutes, and the supernatant was removed to obtain a cell precipitate, namely LGG. Then 0.5mL MnCl2·4H2O solution (0.22mg / mL) and 0.5ml EGCG solution (0.50mg / mL) were added in sequence, and the mixture was vigorously shaken at 1000rpm / min for 5min at 37℃. Centrifuged at 10℃ and 5000rpm for 5 minutes, and the supernatant was removed to obtain a precipitate. The precipitate was washed with HEPES to remove excess reactants to obtain LGG@EGCG-Mn.

[0045] Example 4: Structural characterization and verification of LGG@EGCG-Mn

[0046] The LGG@EGCG-Mn prepared in Examples 1-3 were characterized, and the results were similar, as detailed below:

[0047] The characterization method is as follows: DLS (Mastersizer 3000, UK) was used to measure the size distribution of LGG and LGG@EGCG-Mn. The morphology of the encapsulated probiotics was observed by transmission electron microscopy (Scios, USA). The distribution of surface elements was scanned by field emission transmission electron microscopy (HRTEM) (FEI Tecnai G2F30, USA). For each measurement, a drop of bacterial solution was deposited on a carbon-coated copper grid (200 mesh) for 5 minutes. The sample was then observed after complete drying in air. After labeling the EGCG-Mn layer with rhodamine B, it was characterized by an inverted fluorescence microscope (DFC9000GTI, Leica Microsystems) to visualize the nanocoating on LGG. Natural bacteria LGG and encapsulated bacteria LGG@EGCG-Mn were analyzed using flow cytometry.

[0048] Plotting a growth curve: Using a growth curve instrument (GCA-200, Shineso), the absorbance of LGG, LGG / EGCG prepared in Example 1, and LGG@EGCG-Mn at 600 nm was measured every hour, and a growth curve was plotted.

[0049] Bacterial survival. Plate colony counting method was used. The survival rate of strains under different coating conditions (i.e., LGG, LGG / EGCG, LGG@EGCG-Mn) was evaluated by colony forming units (CFU). All bacterial cells were inoculated in MRS agar plates at different dilutions and incubated at 37°C for 24 h for counting. All data were repeated in at least 2 independent experiments.

[0050] like Figure 1 As shown in A, LGG adsorbs Mn by electrostatic attraction. 2+ , further promoting oxidative polymerization, followed by EGCG self-assembly on the bacterial surface. Within 5 minutes of vigorous shaking, LGG@EGCG-Mn can be quickly formed. Figure 1 C shows that the survival rate and growth rate of LGG coated with EGCG alone are significantly reduced compared with the original LGG. This indicates that the addition of a certain amount of Mn 2+ , significantly improving the survival rate of LGG after encapsulation, achieving unexpected technical effects.

[0051] Depend on Figure 2 As can be seen from A, the DLS results showed that the hydrated size of LGG increased after the nanocoating process, which preliminarily verified the successful encapsulation of LGG cells with the EGCG-Mn layer. In order to visually see the successful encapsulation of a single LGG, the coating was post-functionalized with rhodamine B via non-covalent interactions as described previously. From inverted fluorescence microscopy imaging ( Figure 2 B) and confocal laser scanning microscopy CLSM images ( Figure 2 C) It can be seen that the nanocoating is uniformly formed on a single LGG. Correspondingly, compared with the original LGG, the TEM results ( Figure 2 D) It can be seen that when the addition amounts of EGCG and MnCl2·4H2O were 0.5 mg / mL and 0.22 mg / mL, respectively, a transparent shell (about 400 nm thick) and adhered nanoparticles can be seen on the surface of LGG, which is consistent with the DLS results. Figure 2 E shows that O and Mn elements are evenly distributed around LGG and enriched at the nanoparticle site, which indicates the oxidation and self-assembly behavior of EGCG on the bacterial surface. Flow cytometry analysis results of the fluorescence intensity data of EGCG-Mn labeled rhodamine B ( Figure 2I) showed that the EGCG-Mn coating labeled with rhodamine B was formed on most of the LGG (more than 89%). It is worth emphasizing that the bacterial plate count results and the similar growth curves between the coated bacteria (LGG@EGCG-Mn) and the uncoated bacteria (LGG) proved that the side effects of this rapid encapsulation process on bacterial viability were negligible ( Figure 2 FH).

[0052] In summary, the present application connects EGCG-Mn nanoparticles to the surface of LGG bacteria through a simple "one-pot" reaction. Not only are the reaction steps simple, the conditions mild, and the coverage of the nanocoating high, but more importantly, it has no adverse effect on the bacterial activity of LGG, which is of great significance for the subsequent treatment of UC.

[0053] Example 5: Verification of ROS scavenging and cell protection capabilities of LGG@EGCG-Mn in vitro

[0054] This example verifies the additional antioxidant capacity of EGCG-Mn to LGG. The details are as follows:

[0055] 1. DPPH free radical scavenging activity. LGG and LGG@EGCG-Mn were adjusted to OD 600 1.0 with saline (NaCl, 0.9%) using a multifunctional microplate reader (Varioskan Lux, Thermo Fisher Scientific). Then, the sample was added to a 0.2 mM DPPH ethanol solution (1:2, v / v) and mixed thoroughly. The mixture was allowed to stand at 37 ° C in the dark for 30 minutes. The control group was prepared with deionized water and DPPH alcohol solution (1:2, v / v), and the blank group was prepared with the sample solution and anhydrous ethanol (1:2, v / v). The absorbance was measured at 517 nm.

[0056] The antioxidant activity was calculated using the following formula: DPPH scavenging effect (%) = [1-(A1-A3) / A2] x 100%, where A1 is the absorbance of the test sample, A2 is the control group, and A3 is the blank group. The above test was performed in triplicate.

[0057] 2. Total antioxidant capacity assay (ABTS). According to the kit instructions, first use equal volumes of ABTS solution and oxidant solution to prepare ABTS working stock solution. After storing in the dark at room temperature for 12-16 hours, dilute with PBS to obtain ABTS working solution (A734 = 0.7 ± 0.05, dilution factor is about 30-50 times). Add 200 μL ABTS working solution to each well of a 96-well plate. Then add 10 μL sample solution (LGG or LGG@EGCG-Mn suspension) to the well. Use 10 μL PBS as a blank control. Use 10 μL Trolox solution of different concentrations as a standard. Incubate the 96-well plate at room temperature for 2-6 minutes, and measure the absorbance at 734 nm. The antioxidant capacity of the sample is expressed as Trolox equivalent antioxidant capacity (TEAC).

[0058] 3. Total antioxidant capacity assay (FRAP). Add 180 μL of FRAP working solution to each well of a 96-well plate. Add 5 μL of sample solution (LGG or LGG@EGCG-Mn suspension) to the well. Use 5 μL of 0.15 mM Trolox as a positive control. Use 5 μL of different concentrations of FeSO4·7H2O as a standard. Incubate the 96-well plate at 37°C for 3-5 minutes and measure the absorbance at 593 nm. The total antioxidant capacity is expressed as the concentration of the FeSO4·7H2O standard solution.

[0059] 4. Cell protection ability. LGG and LGG@EGCG-Mn were exposed to different concentrations of H2O2 (0.1, 0.5, 1, 10mM, NaCl 0.9%) for 2h, and then centrifuged (5000rpm, 5min, 4°C) to remove the supernatant. The bacterial precipitate was resuspended in MRS medium, and the bacterial viability was evaluated using the CCK-8 method. LGG and LGG@EGCG-Mn (0.5, 0.25, 0.125mg / mL) were incubated at 1mM H2O2 for 2h; the original LGG without H2O2 incubation was suspended in PBS solution as a blank control group. A growth curve instrument (GCA-200, Shineso) was used to monitor bacterial proliferation within 24 hours and counted on MRS agar plates. The dilution factor of the bacterial solution was 10 6 All data were repeated in at least 2 independent experiments.

[0060] The results of the DPPH free radical scavenging activity experiment showed that: (1) the original LGG had almost no antioxidant activity; (2) the EGCG-Mn modified LGG could effectively scavenge DPPH·, as evidenced by the attenuation of the absorption peak of the DPPH solution at 531 nm ( Figure 3A). At the same time, the scavenging kinetics evaluation experiment found that within 30 minutes, LGG@EGCG-Mn (0.5mg / mL, 0.25mg / mL and 0.125mg / mL) could independently scavenged 80.91%, 82.91% and 76.97% of free radicals (0.2mMDPPH·)( Figure 3 B). Similarly, according to the results of ABTS and FRAP experiments, LGG@EGCG-Mn can effectively change the UV-visible absorbance and exhibit free radical scavenging kinetics and rate in a concentration-dependent manner ( Figure 3 D, F). In summary, LGG@EGCG-Mn exhibits a wide range of free radical scavenging capabilities. Moreover, compared with the PDA-modified probiotics reported in the literature (LGG@PDA), LGG@EGCG-Mn exhibits significantly higher scavenging activity in DPPH free radical scavenging and total antioxidant capacity assay using ABTS and FRAP methods ( Figure 3 A, 3C, 3E).

[0061] In addition, by Figure 3 G shows that EGCG exhibits the ability to inhibit the growth of pathogenic bacteria (Escherichia coli, Staphylococcus aureus) while maintaining the growth of beneficial bacteria (LGG), indicating that it is a more suitable modification option than dopamine (PDA). Figure 3 H clearly showed that after 2 hours of exposure to various exogenous H2O2 (1mM, 0.5mM, 0.1mM) solutions, LGG@EGCG-Mn showed higher bacterial viability than the original LGG. This indicates that due to the antioxidant properties, the cell protection against H2O2-induced oxidative stress was achieved through the modification of LGG by EGCG-Mn. In addition, after incubating LGG coated with different concentrations of EGCG-Mn and uncoated LGG with 1mM H2O2 for 2 hours, the growth curve and bacterial plate count results showed that the system with 0.125mg / mL of EGCG showed a faster growth rate and higher bacterial survival rate under 1mM H2O2 compared with the system with 0.25mg / mL and 0.5mg / mL, providing the best cell protection ( Figure 3 IK).

[0062] Example 6: SA-tK-Ca 2+ Preparation and characterization of hydrogels.

[0063] Preparation method: SA (viscosity 1000–1200 mPa·s), EDC and NHS were added to distilled water at a molar ratio of 2:2:1, 2:1.5:1 and 2:1:1, respectively, and stirred for more than 10 minutes to activate the carboxyl groups on the SA molecules. Then tK was added to the above solution at a molar ratio of 50:1 and 100:1 (SA to tK), respectively, and stirred until the system showed slight cross-linking, and SA-tK semifluid ( Figure 4 A). After the SA-tK semifluid is synthesized, 1 mL of SA-tK semifluid is mixed with 10-20 mg / mL CaCl2 solution at a volume ratio of 1:1 to quickly (stir for about 30 seconds) obtain SA-tK-Ca 2+ Hydrogel.

[0064] Meanwhile, SA-Ca 2+ The hydrogel was used as a control sample for subsequent performance testing. 2+ Hydrogel and SA-Ca 2+ Hydrogels such as Figure 4 As shown in C.

[0065] The semifluid prepared above was characterized and the results were consistent, as detailed below: Fourier transform infrared (FT-IR) spectroscopy results showed that compared with SA, the semifluid had a -1 The presence of an amide bond was found at Figure 4 B), which proves the successful synthesis of SA-tK. The semifluid was refrigerated below -20°C for future use. The microscopic morphology of the hydrogel was observed by scanning electron microscopy (TM 3000, Hitachi), and the diameter distribution diagram was drawn by imageJ software. According to the SEM results, the hydrogel was similar to that of SA-Ca 2+ Compared with hydrogel, SA-tK-Ca 2+ The surface morphology of the hydrogel is uniform and thick, and the pores are significantly smaller ( Figure 4 E). According to quantitative analysis, SA-tK-Ca 2+ Hydrogel and SA-Ca 2+ The average pore sizes on the cross-section of the hydrogel were approximately 171 μm and 381 μm, respectively. Figure 4 F).

[0066] Determination of hydrogel gelation time: SA-tK was added to different concentrations of CaCl2 solution (2-20 mg / mL) and stirred thoroughly. At the same time, different volume ratios of SA:tK and CaCl2 (1:2-2:1) were thoroughly mixed. Then, a 1 mL syringe was used to draw a sufficient amount of the above solution and squeezed out. When the syringe could not squeeze out more liquid, the gelation process was completed and the time was recorded; the experiment was repeated 3 times. The results showed that due to Ca2+ With the increase of concentration, the gelation time decreased from 300 seconds to 10 seconds, but there was no significant difference in the gelation time under different volume ratios (SA-tK: CaCl2). Figure 4 G), which shows that Ca 2+ Concentration is a key factor in the gel formation process.

[0067] Study on the dependence of gel formation process on CaCl2 concentration: First, CaCl2 with different concentrations (0, 5, 10 and 20 mg / mL) and SA-tK semifluid were fully mixed in a volume ratio of 1:1 to obtain hydrogels. The texture of the three hydrogels was observed by naked eyes. Then, the rheometer was used to measure the 2+ The mechanical strength of the hydrogel was studied as follows: CaCl2 and SA-tK semifluid with different concentrations (0, 5, 10 and 20 mg / mL) were injected into the rheometer at a volume ratio of 1:1 at the same time, and the mechanical strength was measured immediately. The test was carried out in vibration mode (fixed strain of 0.5% and frequency of 1 Hz). All measurements were performed at 37°C. The results show that: (1) When the CaCl2 concentration is 0 and 5 mg / mL, the mechanical strength of the hydrogel is relatively low. (2) When the CaCl2 concentration is 10 mg / mL, the mechanical strength of the hydrogel increases; (3) When the CaCl2 concentration is 20 mg / mL, the hydrogel has a relatively hard mechanical strength ( Figure 4 D), therefore, the CaCl2 concentration was selected to be 10-20 mg / mL.

[0068] SA-tK-Ca 2+ The responsiveness of the hydrogel to the environment (pH and ROS). Figure 4 As shown in H, the control sample SA-Ca 2+ The gel structure of the hydrogel was destroyed at pH = 7.4. 2+ The hydrogel swelled and dissolved only when H2O2 was added. 2+ The hydrogel has the potential for colon-targeted applications.

[0069] Example 7: LGG hydrogel encapsulation preparation (LGG@EGCG-Mn@SA-tK-Ca 2+ ) preparation and characterization

[0070] The LGG@EGCG-Mn prepared in Example 1 was mixed with the SA-tK semifluid described in Example 4 and stirred to obtain the semifluid LGG@EGCG-Mn@SA-tK; then an appropriate amount of CaCl2 solution was added and stirred for about 30 seconds to obtain the hydrogel-encapsulated LGG@EGCG-Mn, namely, LGG@EGCG-Mn@SA-tK-CaCl2. 2+ , i.e., LGG hydrogel encapsulation preparation.

[0071] Confocal laser scanning (CLSM) was used: LGG and EGCG-Mn were labeled with FITC and rhodamine B, respectively. Figure 5 As shown in A, LGG@EGCG-Mn has been enclosed in the hydrogel.

[0072] Verification of the ROS / pH dual-responsiveness of LGG hydrogel encapsulation preparations.

[0073] In order to determine the LGG@EGCG-Mn@SA-tK-Ca 2+ The ROS / pH dual response ability of LGG@EGCG-Mn@SA-tK-Ca 2+ and LGG@EGCG-Mn@SA-Ca 2+ Growth curves of SA-tK-Ca in DeMan, Rogosa and Sharpe (MRS) medium at different pH conditions. 2+ Hydrogel and SA-Ca 2+ The hydrogels were resuspended in MRS liquid medium (pH=4, pH=7.4, pH=7.4+1 mM H2O2), and they were incubated at 37°C with shaking at 200 rpm. The OD value was recorded at 600 nm / 1 h by a growth curve analyzer (GCA-200, Shineso).

[0074] like Figure 5 As shown in H and 5I, in the acidic MRS solution (pH = 4), the LGG encapsulated in the two hydrogels did not proliferate significantly. 2+ LGG in hydrogels showed typical growth and stationary phases in neutral MRS (pH = 7.4). 2+ The growth curve of LGG in the hydrogel was slightly delayed in the first two hours, and there was a lower plateau period, indicating that LGG was not completely released. 2+ LGG in the hydrogel was completely released, indicating that it has the function of responding to ROS. 2+ The addition of tK in the hydrogel not only enhances the function of LGG against adverse conditions in the GIT, but also helps the release of LGG under the ROS environment, which lays the foundation for colon-targeted delivery and intestinal colonization after oral probiotic administration.

[0075] Verification of the gastric acid resistance of LGG hydrogel encapsulated preparations.

[0076] Considering that the survival of probiotics in the low pH gastric environment after oral administration is a key challenge, this example studied the survival of natural LGG, LGG@EGCG-Mn and LGG@EGCG-Mn@SA-tK-Ca 2+ Survival rate in simulated gastric fluid (SGF). First, simulated gastric fluid was prepared. Simulated gastric fluid (SGF) was prepared by adding pepsin (0.32 g) and NaCl (0.2 g) to ddH2O (100 mL) and adjusting the pH to 2.0 with HCl solution (0.1 M). SGF was filtered with a membrane filter (0.22 μm) before use. Then, equal amounts of LGG, LGG@EGCG-Mn, and LGG@EGCG-Mn@SA-tK-Ca were cultured in SGF (1 mL). 2+ (10 9 After incubation at 37 °C with gentle shaking (200 rpm) for a predetermined time (0.5, 1, 2 h), LGG, LGG@EGCG-Mn and LGG@EGCG-Mn@SA-tK-Ca 2+ Serial dilutions of the culture medium were plated on MRS agar plates and colony counts were counted after incubation at 37 °C for 36 h.

[0077] In addition, bacteria were stained with DAPI and PI and visualized by an inverted fluorescence microscope (DFC9000GTI, Leica Microsystems). Meanwhile, the relative number of dead bacteria in each group was quantified by flow cytometry.

[0078] like Figure 5 As shown in 5C, 5D, and 5E, both encapsulated LGGs showed stronger SGF tolerance than original LGG, and this was more obvious over time. After exposure to SGF for 1 and 2 hours, the survival rate of LGG@EGCG-Mn was slightly higher than that of original LGG, indicating that the metal-polyphenol network had a certain cytoprotective effect. After the incubation time was extended from 1 hour to 2 hours, only a small part of the original LGG cells survived, while LGG@EGCG-Mn@SA-tk-Ca 2+ The number of surviving bacteria in the group was significantly higher than that in the other two groups ( Figure 5 B, 5E). This preliminarily proves that the LGG hydrogel encapsulation preparation described in the present application can achieve the desired protective effect.

[0079] In addition, after SGF incubation, PI (red, dead bacteria) and DAPI (blue, all bacteria) were used to distinguish dead bacteria ( Figure 5 G). Obviously, gastric acid is more lethal to naked LGG. Almost all LGG cells and LGG@EGCG-Mn died after one hour of SGF incubation. In contrast, LGG@EGCG-Mn@SA-tk-Ca2+ Only a relatively small number of dead bacteria appeared in the group, further confirming the protective effect of the LGG hydrogel encapsulation formulation.

[0080] At the same time, after SGF incubation, the bacteria stained with PI were analyzed by flow cytometry to detect the fluorescence intensity data of dead bacteria labeled with PI. The results are as follows Figure 5 F. In the LGG group and LGG@EGCG-Mn group, the bacterial mortality rate was as high as 99%, while in the LGG@EGCG-Mn@SA-tk-Ca 2+ The mortality rate in the group was only 28.4%, which verified the reliability of the above conclusions.

[0081] Evaluation of the enhanced mucosal adhesion of encapsulated probiotics in vivo.

[0082] In order to determine the 2+ The intestinal retention time of the mouse was monitored by in vivo imaging system (IVIS). Specifically, C57BL / 6N mice were randomly divided into 3 groups (n=3 in each group) and given equal doses (10 9 CFU) of EGCG-Mn, LGG@EGCG-Mn and LGG@EGCG-Mn@SA-tK-Ca 2+ EGCG-Mn was labeled with rhodamine B, and fluorescence images were taken using the IVIS system at 1h, 5h, 9h, and 24h after administration. The mice were euthanized at 24h under deep anesthesia. The intestines were immediately harvested and observed under IVIS. The schematic diagram of the experimental operation is shown in Figure 6 As shown in A. Figure 6 B It can be clearly seen that after 9 hours of oral administration, the fluorescence intensity of the EGCG-Mn group was significantly lower than that of the other two groups, indicating that the nanoparticle administration itself has the lowest ability to retain particles in the gastrointestinal tract. In addition, the fluorescence signal of the LGG@EGCG-Mn group began to weaken at 24 hours. At the same time, the fluorescence intensity of the LGG@EGCG-Mn@SA-tK-Ca 2+ Relatively strong fluorescence could still be detected in the stomach of the group, indicating that this group had the highest gastrointestinal retention rate ( Figure 6 C).

[0083] To evaluate targeted adhesion to inflamed lesions in the colon, LGG was labeled with Cy5.5-NHS to confirm the release of SA-tK-Ca in response to colonic inflamed lesions. 2+ Hydrogel-encapsulated LGG. Mice were continuously administered 2.5% DSS to induce UC, and then UC mice were gavaged with 0.1 mL of LGG hydrogel encapsulated formulation. The time to colon was tracked using IVIS ( Figure 6D). In LGG@EGCG-Mn@SA-tK-Ca 2+ 4h and 8h after administration, mice were killed, and GIT was collected and observed under IVIS. The colon of mice killed 8h after administration was frozen and sectioned and observed under a fluorescence microscope. As expected, according to the frozen section results ( Figure 6 E) shows that there is a large amount of Cy5.5-labeled LGG in the colon of UC mice. In summary, colon-targeted hydrogel plays a key role in enhancing the adhesion of LGG and EGCG-Mn to the inflammatory area.

[0084] Example 8: LGG@EGCG-Mn@SA-tK-Ca 2+ Preventive effect on DSS-induced colitis.

[0085] This example studies LGG@EGCG-Mn@SA-tK-Ca 2+ The preventive effect in the DSS-induced acute colitis model, such as Figure 7 The specific operation was as follows: the mice were randomly divided into 6 groups: normal group, model group, LGG group, LGG@EGCG-Mn group, LGG@SA-tK-Ca 2+ Group and LGG@EGCG-Mn@SA-tK-Ca 2+ The experimental period was 14 days. The mice in the normal group had free drinking water and were gavaged with 0.1 mL PBS every day. The mice in the other five groups had free drinking water in the first 6 days, and were given 2.5% DSS on days 7-14 to induce colitis. Among them, the model group was gavaged with 0.1 mL PBS every day like the normal group, while the LGG group, LGG@EGCG-Mn group, and LGG@SA-tK-Ca 2+ Group and LGG@EGCG-Mn@SA-tK-Ca 2+ The groups were gavaged with 0.1 mL of the corresponding drug every day (the gavage bacterial dose was 10 9 CFU, SA-tK-Ca 2+ The dose was approximately equal to 45 mg / kg.) The mice were observed for body weight, fecal characteristics, fur color, activity, and mental state at 6:00 p.m. every day. On the last day of treatment, the mice were fasted but not dehydrated. The day after treatment was completed, the mice were euthanized by cervical dislocation, and the colon and spleen were separated, measured and weighed. The blood was centrifuged to obtain serum. The colon was cut open and fixed with 4% paraformaldehyde for histological analysis. The feces and the rest of the colon were frozen at -80°C for further analysis.

[0086] The typical symptoms of acute colitis are characterized by indicators such as body weight, disease activity index (DAI), spleen index and colon length, and histopathological studies were performed. Specifically: colon samples were fixed with 4% paraformaldehyde and embedded in paraffin, then cut into 4 μm thick sections and processed for H&E staining and AB-PAS staining. The staining results were scanned with an optical microscope (EclipseE100, Nikon). According to the results of histopathological analysis, a scoring system was used to evaluate the degree of colon damage (Table S1). Figure 7 As shown in BF, mice treated with DSS showed significant weight loss, shorter colon length, high DAI score, and high spleen index score, which fully demonstrated that the mouse UC model was successfully established. 2+ and LGG@EGCG-Mn@SA-tK-Ca 2+ After addition of LGG@EGCG-Mn@SA-tK-Ca 2+ The treatment group was superior to the other 4 groups in maintaining colon length, weight restoration, DAI reduction, and spleen index reduction, indicating the effectiveness of EGCG-Mn-modified LGG combined with intestinal hydrogel for the prevention of UC.

[0087] H&E staining was used for histological analysis of colon samples. Specifically, fixed colon tissue sections were immersed in Triton-X100 and then immersed in citrate buffer to recover antigens. After sealing with H2O2 and serum, they were incubated with ZO-1 and Occludin-1 primary antibodies overnight. Next, the sections were washed with PBS and incubated with horseradish peroxidase (HRP)-labeled secondary antibodies, stained with DAPI, and observed using an inverted fluorescence microscope.

[0088] like Figure 7 As shown in G, the normal mouse group had a well-shaped intestine characterized by intact colonic epithelium, tightly arranged intestinal crypts, and sufficient goblet cells. In contrast, the intestine of the model group depicted severely damaged mucosa, showing branching, atrophy, and irregular intestinal crypts, lymphocyte infiltration, and a decrease in the number of goblet cells. The intestinal conditions observed in the LGG and LGG@EGCG-Mn groups improved slightly, indicating that the two had limited effects on alleviating these symptoms. At the same time, in the LGG@SA-tK-Ca 2+ Group and LGG@EGCG-Mn@SA-tK-Ca 2+ In the LGG@EGCG-Mn group, normal crypt surfaces and intact goblet cells were observed, indicating that the two have a significant effect on alleviating the aforementioned symptoms. It should be emphasized that the LGG@EGCG-Mn group has a better symptom relief effect than the LGG group alone.

[0089] In addition, tight junction proteins play a vital role in maintaining the integrity of colon tissue. Therefore, this application detects the integrity of the intestinal mucosal barrier function and structure of mice by measuring the levels of two tight junction proteins in mouse colon tissue. Immunofluorescence staining results ( Figure 7 I, 7J) showed that the levels of two tight junction proteins (occludin and ZO-1) in the colon tissue of normal mice were relatively high, indicating the integrity of the colon tissue and intestinal mucosal barrier. However, after DSS treatment, the expression of tight junction proteins ZO-1 and occludin was significantly reduced. After LGG treatment, the levels of occludin and ZO-1 were restored; but compared with the normal group, the degree of recovery was not obvious. After treatment in the last three groups, the levels of the two tight junction proteins were significantly restored.

[0090] Dysregulated immune responses are associated with the pathogenesis and progression of UC. Real-time quantitative PCR assay. Total RNA was extracted from colon tissue using a kit (AU1001S, BioTeke Corporation) and reverse transcribed into complementary DNA (cDNA). Primer sequences are shown in Table S3. RT qPCR was performed according to the kit instructions. The data were analyzed using the 2-ΔΔCT method, with Gapdh as the housekeeping gene. The thermal cycler program was as follows: pre-denaturation (95°C, 30 seconds), denaturation (95°C, 10 seconds), annealing (56°C, 30 seconds), and extension (72°C, 30 seconds).

[0091] The present application measured the colonic mRNA expression of genes encoding the proinflammatory cytokines IL-1β, TNF-α, and IL-6. As expected, LGG@EGCG-Mn@SA-tK-Ca 2+ Treatment significantly reduced the expression of all three ( Figure 7 H).

[0092] In summary, the induction of DSS may lead to the destruction of intestinal mucosal barrier and colon tissue integrity, thus leading to the occurrence of UC; while LGG@EGCG-Mn@SA-tK-Ca 2+ The treatment showed excellent protective effects on improving inflammatory factors in the colon barrier and intestinal mucosal barrier.

[0093] Example 9: LGG@EGCG-Mn@SA-tK-Ca 2+ Study on the mechanism of alleviating DSS-induced colitis

[0094] This example studies LGG@EGCG-Mn@SA-tK-Ca 2+ The effect on the improvement of the intestinal microbial barrier in mice is to further reveal its mechanism.

[0095] First, intestinal microbiota analysis was performed. Total genomic DNA was extracted from fecal samples according to the instructions (TGuide S96Soil / Fecal DNAKit, Tiangen). The bacterial 16S rDNA V3-V4 region was then amplified and sequenced using primer 338F_806R, and the forward and reverse 16S primers were tail sequenced using sample-specific Illumina index sequences. The sequencing platform was Illumina NovaSeq. Microbial diversity was analyzed using the biocloud platform. Operational taxonomic units (OTUs) are identical markers that are artificially set for a certain taxonomic group for ease of analysis.

[0096] like Figure 8 As shown in A, the use of DSS significantly reduced the abundance of OUT, while LGG@EGCG-Mn@SA-tK-Ca 2+ The use of LGG@EGCG-Mn@SA-tK-Ca significantly reversed this change, which represents an increase in the abundance of intestinal flora. In addition, fecal sample analysis showed that compared with the model group, LGG@EGCG-Mn@SA-tK-Ca 2+ The administration of also significantly increased the α diversity of the intestinal microbiota, while the other three groups had no significant improvement ( Figure 8 B). The extent and magnitude of structural variation between samples is mainly reflected by the sample distance, as shown by β diversity. Figure 8 The results of principal coordinate analysis (PcoA) and nonmetric multidimensional scaling (NMDS) analysis (C and 8D) showed that the DSS group was significantly separated from the normal group; while among the four drug-treated groups, LGG@EGCG-Mn@SA-tK-Ca 2+ The intestinal flora composition of DSS-induced colitis mice treated with the LGG@EGCG-Mn@SA-tK-Ca 2+ administration reshaped and regulated the intestinal flora of mice. In addition, the results of microbiome composition analysis ( Figure 8 E) shows that DSS induction reduced the relative abundance of Bacteroidota and increased the proportion of Firmicutes compared with the normal group. Interestingly, this change was not observed in the LGG@EGCG-Mn@SA-tK-Ca 2+ After treatment, the ratio of Bacteroidetes to Firmicutes was reversed, resulting in a ratio close to that of the normal group. 2+ In the group, an increase in the content of verrucomicrobia was observed.

[0097] Next, heat map analysis was used to visualize the detailed changes in gut microbiota ( Figure 8 F). Figure 8F shows the average relative abundance of the 30 most abundant taxa at the family level. 2+ The treatment significantly increased the relative abundance of beneficial bacteria, such as Bacteroidetes, Akkermansiaceae, Lachnospiraceae, and Ruminococcaceae, while this treatment reduced the occurrence of harmful bacteria, such as Clostridia-UCG-014.

[0098] In addition, linear discriminant analysis (LDA) effect size (LEfSe) analysis was performed with an LDA score of 2 to identify significant biomarkers; the normal group and LGG@EGCG-Mn@SA-tK-Ca 2+ The treatment group was mainly characterized by beneficial bacteria. It is worth noting that LGG@EGCG-Mn@SA-tK-Ca 2+ group contained Lactobacillus rhamnosus, which distinguished it from the other two groups and verified the successful colonization of LGG ( Figure 8 G). Finally, we analyzed the composition of the gut microbiota at the genus and species levels ( Figure 8 H). From the sequencing results, the abundance of Clostridia-UCG-014 reported in the colitis model group increased significantly, and this phenomenon was confirmed by using LGG@EGCG-Mn@SA-tK-Ca 2+ Similarly, the proportions of Lacnospiraceae and AKKermansia muciniphila returned to normal levels ( Figure 8 I).

[0099] Inspired by the results of the gut microbiome analysis, we then studied the fecal SCFAs levels in different groups. The specific operation was as follows: the fecal samples were mixed with 500 μL of water, 300 μL of 50% sulfuric acid was added, 50 μL of internal standard solution (cyclohexanone, 500 mg / L) and 1 mL of ether were added for 1 min, and centrifuged at 12 000 rpm for 10 min at 4 °C. The supernatant was extracted for GC-MS detection (GCMS QP2010-Ultra, SHIMADZU), and the results are detailed in Figure 8 J. As Figure 8 As shown in J, the concentrations of acetate, propionate, butyrate, caproate, valerate, and branched-chain SCFAs isobutyrate and isovalerate in the colitis model group were significantly lower than those in the normal group. However, after supplementation with different pharmaceutical preparations containing LGG, they could gradually return to normal levels. In general, the results of this example provide more evidence that LGG@EGCG-Mn@SA-tK-Ca 2+ The composition of the intestinal microbiota can be actively controlled and the metabolism of SCFAs can be promoted.

[0100] In summary, the Lactobacillus rhamnosus (LGG) hydrogel encapsulation preparation provided in the present application overcomes the technical problems in the prior art that LGG cannot reach the colon in sufficient quantities and is difficult to survive stably, produces unexpected technical effects, and is expected to be used for the treatment of ulcerative colitis, with important social value and huge economic value.

Claims

1. Intestinal targeted pH-ROS dual-responsive hydrogel encapsulation system, characterized by: The hydrogel encapsulation system is a sodium alginate (SA)-based hydrogel modified by ketothiol (TK) and calcium ions, and the hydrogel is used for intestinal probiotics.

2. The pH-ROS dual-responsive hydrogel encapsulation system according to claim 1, characterized in that: The intestinal probiotics are Lactobacillus rhamnosus GG (LGG).

3. The pH-ROS dual-responsive hydrogel encapsulation system according to claim 1, characterized in that: The preparation method is as follows: (1) SA, EDC and NHS are added to water and stirred to activate the carboxyl groups on the SA molecules to obtain solution I; (2) an appropriate amount of TK is added to solution I and stirred until the system is slightly cross-linked, thus obtaining a SA-tK semifluid; (3) the SA-tK semifluid is mixed with an appropriate amount of 10-20 mg / mL CaCl2 solution and stirred for 30 s to obtain SA-tK-CaCl2. 2+ Hydrogel.

4. The pH-ROS dual-responsive hydrogel encapsulation system according to claim 3, characterized in that: In step (1), the molar ratio of SA, EDC and NHS is 2:1:1 to 2:2:1; in step (2), the molar ratio of SA to TK is 50:1 to 100:1; In step (3), the volume of the SA-tK semifluid is not greater than the volume of the CaCl2 solution.

5. A Lactobacillus rhamnosus (LGG) hydrogel encapsulated preparation for the treatment of ulcerative colitis, characterized in that: The LGG hydrogel encapsulation preparation is composed of LGG and a hydrogel encapsulating LGG; the hydrogel is the pH-ROS dual-responsive hydrogel according to any one of claims 1 to 4; wherein the unit volume of the hydrogel contains 10 8 -10 9 CFU of LGG.

6. The LGG hydrogel encapsulation preparation according to claim 5, characterized in that: The LGG is LGG modified with EGCG-Mn nanoparticles, namely LGG@EGCG-Mn; wherein the amount of EGCG-Mn is not less than three times the amount of LGG.

7. The method for preparing the LGG hydrogel encapsulated preparation according to claim 6, characterized in that: The following steps are involved: (1) LGG modified with EGCG-Mn nanoparticles, i.e., LGG@EGCG-Mn, was prepared by in situ deposition; (2) LGG@EGCG-Mn was mixed and stirred with SA-TK semifluid to obtain LGG@EGCG-Mn@SA-tK; (3) Finally, an appropriate amount of CaCl2 solution was added and stirred for 30 seconds to obtain hydrogel-encapsulated LGG@EGCG-Mn, i.e., LGG@EGCG-Mn@SA-tK-CaCl2. 2+ , i.e., LGG hydrogel encapsulation preparation.

8. The preparation method according to claim 7, characterized in that: The step (1) specifically comprises: (a) incubating LGG in MRS culture medium at 37° C. for 12 hours to obtain an LGG suspension; (b) taking a volume of V of the LGG suspension, centrifuging at 4-20° C. to remove the supernatant, to obtain a cell precipitate, i.e., LGG; (c) sequentially adding a MnCl2·4H2O solution and an EGCG solution, both with a volume of V / 2, to the LGG, shaking vigorously at 37° C., and centrifuging at 4-20° C. to remove the supernatant to obtain a precipitate, i.e., LGG@EGCG-Mn; wherein the concentration of the MnCl2·4H2O solution is 0.055-0.22 mg / mL, and the concentration of the EGCG solution is 0.125-0.500 mg / mL.

9. The preparation method according to claim 8, characterized in that: The centrifugation in step (b) is as follows: centrifugation at 5000 rpm for 5 minutes; the cell pellet is washed 3 times with HEPES; the vigorous shaking in step (c) is as follows: oscillation at 600-1000 rpm / min for 5 minutes, and the centrifugation is as follows: centrifugation at 5000 rpm for 5 minutes; the pellet is washed with HEPES.

10. Use of the Lactobacillus rhamnosus (LGG) hydrogel encapsulated preparation according to claim 5 or 6 in the treatment of ulcerative colitis.

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