Hyaluronic acid-based nanogel with intestinal targeting and inflammation responsiveness as well as preparation method and application of hyaluronic acid-based nanogel

Abstract

The invention relates to hyaluronic acid-based nanogel with intestinal targeting and inflammation responsiveness as well as a preparation method and application of the hyaluronic acid-based nanogel, and discloses multifunctional composite nanogel based on hyaluronic acid as well as a preparation method and application of the multifunctional composite nanogel. The preparation method comprises the following steps: loading galangin into bovine serum albumin to prepare BSA-GA nanoparticles with antioxidant and anti-inflammatory activity; 2, synthesizing a multifunctional molecule Se-MHA which is targeted to M2 macrophages and responds to ROS degradation on the basis of hyaluronic acid; 3, BSA-GA nano-particles with the same solution concentration are used as anti-inflammatory functional components to be mixed with Se-MHA to prepare Se-MHA / BG nano-gel, model probiotics are subjected to single-cell modification, probiotic-loaded nano-gel EcN-Se-MHA / BG is prepared, the nano-gel has specific binding and responding capacity on the colitis disease part, colonization of EcN on the colon part is promoted, and the anti-inflammatory effect is achieved. The IBD disease is effectively relieved.

Description

Technical Field

[0001] The present application relates to a nanogel material, in particular to a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness, and also to a preparation method thereof, and the application of the hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness as a drug carrier. Background Art

[0002] Inflammatory bowel disease (IBD) is a non-infectious, chronic intestinal inflammatory disease, primarily characterized by ulcerative colitis (UC). Its pathological hallmarks include chronic inflammation of the intestinal mucosa, impaired epithelial barrier function, elevated levels of reactive oxygen species (ROS), and an imbalance in the intestinal microbiome. These interplay leads to a vicious cycle in IBD treatment. Currently, probiotic therapy has garnered significant attention due to its favorable biosafety profile. It improves IBD symptoms by positively regulating the intestinal microbiome, enhancing the intestinal mucosal barrier, and competitively inhibiting or eliminating pathogens. However, due to the complex environment of gastrointestinal inflammation, probiotics are easily inactivated after administration, making it difficult for them to effectively colonize and exert their effects at the site of inflammation. Summary of the Invention

[0003] In response to the main pathological characteristics of IBD, this paper designs a multifunctional composite nanogel (Se-MHA / BG) with hyaluronic acid (HA) as the framework, and applies it to the modification of the model probiotic Escherichia coli Nissle 1917 (EcN), aiming to improve the delivery efficiency of probiotics in the intestine, while targeting inflammatory sites to enhance the therapeutic effect of probiotics on multiple pathological characteristics of IBD, in order to provide a sustainable solution for the long-term treatment of IBD.

[0004] In order to achieve the above technical objectives, the present invention provides a method for preparing hyaluronic acid-based nanogels with intestinal targeting and inflammatory responsiveness. In the first step, galangin GA was loaded into bovine serum albumin (BSA) to prepare BSA-GA nanoparticles with antioxidant and anti-inflammatory activities; In the second step, a multifunctional molecule Se-MHA was synthesized based on hyaluronic acid (HA) to target M2 macrophages and respond to ROS degradation; In the third step, BSA-GA nanoparticles (BG) with the same solution concentration as the anti-inflammatory functional component were mixed with Se-MHA at a volume ratio of 4:1 to prepare Se-MHA / BG nanogels; Finally, Se-MHA / BG nanogel was used to modify the model probiotic Escherichia coli Nissle 1917 (EcN) by single cells to prepare the probiotic-loaded nanogel EcN@Se-MHA / BG.

[0005] Preferably, the present application provides a preparation method of hyaluronic acid-based nanogel with intestinal targeting and inflammation responsiveness, comprising: Step 1, preparing BSA-GA nanoparticles BG by loading the hydrophobic flavonoid galangin GA with anti-inflammatory activity into bovine serum albumin BSA through ethanol desolvation method (adding 0-100 μM galangin GA anhydrous ethanol solution into bovine serum albumin BSA slowly and stirring for 2 hours under light-proof conditions, controlling the final concentration of ethanol in the solution within 2%, and removing ethanol by rotary evaporation at 35°C for 10 minutes and supplementing the lost volume with double distilled water) and pH shift method (adjusting pH12 with 50 μM BSA, adding 0-100 μM galangin GA solution into bovine serum albumin BSA slowly, stirring for 2 hours under light-proof conditions, and finally adjusting pH7.4); Step 2: First, the mannose Man group is modified to prepare MHA by modifying the mannose Man group of hyaluronic acid HA, and further cross-linked to form a continuous network Se-MHA by using diselenium bond (Se-Se); this design has double advantages: on the one hand, hyaluronic acid HA can specifically bind to CD44 receptors at the inflamed site of the intestine, and the modification of the mannose group endows hyaluronic acid HA with the ability to target M2 macrophages; on the other hand, the diselenium bond is oxidized to selenic acid and broken in the intestinal inflammatory environment, thereby helping the nanogel to dissociate at the inflammation site and playing a role in removing ROS in the microenvironment.

[0006] Step 3: adsorbing BSA-GA nanoparticles on the surface of Se-MHA by electrostatic interaction to form Se-MHA / BG nanogel; Step 4, using the nanogel described in step 3 to modify model probiotic EcN to prepare EcN@Se-MHA / BG.

[0007] Further preferably, Regarding the preparation of BSA-GA nanoparticles, it includes: Step 101, dissolving bovine serum albumin BSA in double distilled water and stirring for 2 hours, and storing overnight at 4°C to fully hydrate, to prepare 50 μM bovine serum albumin BSA working solution; Step 102, slowly adding 0-100 μM galangin GA anhydrous ethanol solution into the bovine serum albumin BSA solution obtained in step 101, and stirring for 2 hours under light-proof conditions to control the final concentration of ethanol in the solution within 2%; Step 103, removing ethanol by rotary evaporation of the bovine serum albumin BSA solution obtained in step 102 at 35°C for 10 minutes, and supplementing the lost volume with double distilled water; Step 104: The bovine serum albumin (BSA) solution obtained in step 103 is adjusted to pH 12 using 2M NaOH, and 0-100 μM galangin (GA) is dissolved in double-distilled water at the same pH value to achieve a final concentration controlled within 2%. Similarly, the aqueous solution containing galangin (GA) is slowly added dropwise to the bovine serum albumin (BSA) solution and stirred for 2 hours in the dark. Finally, the pH value is accurately adjusted back to 7.4.

[0008] The preparation of Se-MHA / BG nanogel includes: Step 201, dissolving 68.5 mg of polybutylene adipate (PBA) and 90 mg of mannose in 20 mL of methanol, and reacting at room temperature for 6 hours to prepare an intermediate of mannose phenylboronic acid; Step 202, dissolving 200 mg of hyaluronic acid (HA) in 20 mL of deionized water, and slowly adding 155 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 86.3 mg of activated methoxypolyethylene glycol (NHS) to the solution, and stirring at room temperature for 60 minutes at pH 5 to activate the carboxyl groups on the hyaluronic acid (HA); Step 203, then continuing to add phenylboronic acid mannose to the solution and adjusting the solution to pH 8, stirring for 24 hours to obtain MHA. In order to remove unreacted molecules, the reaction product is dialyzed three times and collected by freeze drying; Step 204, completing the diselenide bridging of MHA, dissolving 200 mg of MHA in 20 mL of deionized water, and activating the carboxyl groups according to step 202, adding 159 mg of selenocystamine hydrochloride to the above mixture, reacting in the dark for 24 hours, dialyzing the reaction product three times, and lyophilizing to obtain Se-MHA powder; 5 mg / mL Se-MHA solution and 5 mg / mL BSA-GA (BG) solution were prepared separately and thoroughly mixed at a volume ratio of 4:1, so that the nanoparticles BG were adsorbed on the surface of the Se-MHA network through electrostatic action to form Se-MHA / BG nanogel.

[0009] A hyaluronic acid-based nanogel with intestinal targeting and inflammation responsiveness is obtained by the preparation method. The nanogel has specific binding and responsiveness to colonic inflammation sites, promotes EcN colonization in the colon, effectively alleviates IBD symptoms, increases the diversity of microbial composition in the inflamed intestine, and regulates the ratio of Bacteroidetes to Firmicutes. The abundance of beneficial bacteria Lactobacillus is increased, and the abundance of pathogenic bacteria such as Bacteroides is reduced. It helps to increase the levels of various metabolites such as uridine and 2'-deoxyuridine in the intestinal purine metabolism and pyrimidine metabolism pathways, thereby improving inflammation.

[0010] The present invention relates to an application of a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness. The above-mentioned hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness is used to modify the single cell of a model probiotic to prepare the nanogel EcN@Se-MHA / BG loaded with the model probiotic Escherichia coli Nissle 1917 (EcN) as a carrier for oral probiotics. Beneficial effects

[0011] The present invention designs and synthesizes a multifunctional nanogel based on a protein / polysaccharide electrostatic composite system. The gel has intestinal targeting and inflammatory responsiveness, and has the advantages of simple preparation method, high biocompatibility, and low toxic and side effects.

[0012] The nanogel prepared by the present invention is used as a coating material for the model probiotic EcN, which effectively improves the targeted colonization ability of EcN in the intestines of IBD patients. The introduced diselenide bonds and mannose groups give it the ability to target M2 macrophages and respond to and clear ROS. The introduction of the anti-inflammatory active ingredient GA further slows down the occurrence of intestinal inflammation, reshapes intestinal homeostasis, and improves long-term therapeutic effects.

[0013] The nanogel provided by the present invention can be used as a carrier for oral probiotics, is easy to use, and helps to improve patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is the average particle size and PDI of BSA-GA nanoparticles treated by the desolvation method and pH shift method of the present application; Figure 2 Figure 2 shows the uptake of BSA-GAd and BSA-GApH in RAW264.7 cells after 4 hours of incubation at 37°C. Bright field (BF) confocal laser scanning microscopy images of DAPI-stained cell nuclei (blue), FITC-labeled nanoparticles (green), and their fusion layer (A). Cellular uptake kinetics (B), saturation cellular uptake value (Ymax), and uptake rate constant k (C) are shown. Figure 3 The cellular antioxidant properties and inflammation-alleviating effects of BSA-GA nanoparticles and their components treated with the solvent removal and pH shift methods described in this application are shown. ABTS and DPPH scavenging abilities (A), cytotoxicity against RAW264.7 cells (B), effects on ROS levels in H2O2-treated RAW264.7 cells (C), LDH release (D), and secretion of pro-inflammatory cytokines (TNFα, IL-1β, IL-6) and anti-inflammatory cytokines (IL-10) (EH). Figure 4Schematic diagram of the combination of Se-MHA and BG of the present application (A) and SEM microstructure images of HA, BG, Se-MHA and Se-MHA / BG (B); Figure 5 The scattered light intensity change curves of Se-MHA / BG and HA / BG of the present application at pH 7.4-1 (A) and 0-400 μM H2O2 concentration (B); Figure 6 Schematic diagram of the formation and microstructure of EcN@Se-MHA / BG of this application (A), particle size distribution and zeta potential difference of EcN and EcN@Se-MHA / BG (B and C), pH response and H2O2 responsive phase behavior of EcN@Se-MHA / BG and EcN@HA / BG (D); Figure 7 are the growth curves of EcN, EcN@HA / BG, and EcN@Se-MHA / BG of the present application; Figure 8 Schematic diagram of the responsive phase behavior of EcN@Se-MHA / BG in simulated gastrointestinal digestion and mucus layer (A), the survival of probiotics of EcN, EcN@HA / BG, and EcN@Se-MHA / BG in simulated gastrointestinal digestion (B), and the microscopic morphology and bacterial plating in simulated gastrointestinal digestion (C); Figure 9 Trajectory analysis of the EcN and EcN@Se-MHA / BG in a simulated mucus layer (A), average movement speed (B), and distance from the initial position (C), degradation of EcN@Se-MHA / BG in a simulated mucus layer (D), and removal of H2O2 (E); Figure 10 Schematic diagram of the establishment and administration of the DSS-induced colitis mouse model for IVIS imaging in this application (A), fluorescence distribution images of EcN@HA / BG and EcN@Se-MHA / BG in the intestines of healthy mice and colitis mice 6 hours after administration after Cy5.5 staining (B and C), and the total radiation efficiency in the small intestine and colon (D and E); Figure 11 is the co-localization fluorescence analysis of Se-MHA / BG and HA / BG with CD44 and CD206 receptors in the inflamed colon of the present application; Figure 12 Quantitative analysis of Escherichia coli in the intestinal contents of mice after oral gavage with EcN, EcN@HA / BG, and EcN@Se-MHA / BG of this application (A) and representative images of colonies on diluted eosin-methylene blue plates (B); Figure 13Schematic diagram of the DSS-induced colitis mouse treatment of the present application (A), changes in body weight and disease activity index (DAI) of mice in different treatment groups over 12 days (B and C), images of colon and spleen tissues in different prevention groups (D), colon length and spleen index (E and F); Figure 14 Figures 1 and 2 show hematoxylin & eosin (H&E) staining, AB-PAS staining, and TUNEL staining images (A) and histochemical scores (B) of intestinal tissue sections of mice in different treatment groups of the present application, and immunofluorescence staining images (C) and semi-quantitative analysis (D) of mucin and tight junction proteins (ZO-1 and Occludin) in intestinal tissues of mice in different treatment groups. Figure 15 Immunofluorescence images of CD86 and CD206 receptors in colon tissue sections of different treatment groups of the present application (A) and changes in the ratio of M2 to M1 macrophages (B); Figure 16 The levels of inflammatory markers TNF-α (A), IL-6 (B), IL-1β (C) and IL-10 (D), the level of oxidative stress marker MDA (E) and the activities of related antioxidant enzymes MPO, GSH-px and SOD (FH) in colon tissue of the present application; Figure 17 Schematic diagram of the prevention of DSS-induced colitis in mice of the present application (A), changes in body weight and disease activity index (DAI) of mice in different treatment groups over 12 days (B and C), images of colon and spleen tissues in different treatment groups (D), colon length and spleen index (E and F); Figure 18 Figures 1 and 2 show hematoxylin & eosin (H&E) staining, AB-PAS staining, TUNEL staining, and histochemical scoring of intestinal tissue sections of mice in different treatment groups of the present application (A and B), as well as images of immunofluorescence staining of CD86 and CD206 receptors and changes in the proportion of M2 and M1 macrophage phenotypes (C and D). Figure 19 Comparison of the α-diversity (Shannon index, Simpson index, and Chao1 index) of the intestinal microbiota of mice in the DSS-induced colitis treatment experiment (AC) and prevention experiment (DF) of this application; Figure 20 The relative abundance of the intestinal microbiota of mice at the phylum level and the relative abundance of Proteobacteria in the DSS-induced colitis treatment experiment (A and B) and prevention experiment (C and D) of this application are shown; Figure 21 The relative contents of some differential metabolites that changed significantly in the DSS-induced mouse colitis treatment experiment (AD) and prevention experiment (EH) of the present application. DETAILED DESCRIPTION

[0015] For the purpose of facilitating the understanding of the present application, the present application will be described in more detail below in conjunction with the accompanying drawings and specific embodiments.

[0016] Unless otherwise defined, all technical and scientific terms used in the present specification are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the present application belongs. The terminology used in the present specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. As used in the present specification, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0017] Furthermore, the technical features involved in the different embodiments of the present application described below can be combined with each other as long as there is no conflict.

[0018] A preparation method of hyaluronic acid-based nanogel with intestinal targeting and inflammation responsiveness, Firstly, the GA was loaded into BSA to prepare BSA-GA nanoparticles with antioxidant and anti-inflammatory activities; Secondly, a multifunctional molecule Se-MHA targeting M2 macrophages and degrading in response to ROS was synthesized based on HA; Thirdly, the BSA-GA nanoparticles BG with the same solution concentration were mixed with Se-MHA as anti-inflammatory functional components at a volume ratio of 4:1 to prepare Se-MHA / BG nanogel; Finally, the Se-MHA / BG nanogel was modified with model probiotic Escherichia coli Nissle 1917 (EcN) to prepare probiotic-loaded nanogel EcN@Se-MHA / BG. EMBODIMENT

[0019] A preparation method of hyaluronic acid-based nanogel with intestinal targeting and inflammation responsiveness, Step 1: BSA-GA nanoparticles (BG) were prepared by loading the hydrophobic flavonoid galangin GA with anti-inflammatory activity into bovine serum albumin (BSA) using an ethanol desolvation method (an anhydrous ethanol solution containing 0-100 μM galangin GA was slowly added dropwise to a bovine serum albumin (BSA) solution and stirred for 2 hours in the dark. The final ethanol concentration was controlled within 2%. The ethanol was removed by rotary evaporation at 35°C for 10 minutes, and the lost volume was supplemented with double-distilled water) and a pH shift method (50 μM BSA was used to adjust the pH to 12, and the 0-100 μM galangin GA solution was slowly added dropwise to the bovine serum albumin (BSA), stirred for 2 hours in the dark, and finally adjusted to pH 7.4). Figure 1 As shown, the average particle size and PDI of BSA-GA nanoparticles treated by desolvation method and pH shift method; Step 2: First, the HA molecule is modified with a mannose group to prepare MHA, and then further cross-linked using a diselenide bond (Se-Se) to form a continuous network Se-MHA, such as Figure 4 Figure 2 shows a schematic diagram of the binding of Se-MHA to BG (A) and SEM microstructural images of HA, BG, Se-MHA, and Se-MHA / BG (B). This design has dual advantages: on the one hand, HA can specifically bind to the CD44 receptor in intestinal inflammation sites, and the modification of the mannose group gives HA the ability to target M2 macrophages; on the other hand, the diselenide bond is oxidized by ROS to selenic acid and breaks in the intestinal inflammatory environment, thereby facilitating the dissociation of the nanogel at the inflammatory site and playing a role in clearing ROS in the microenvironment.

[0020] Step 3: BSA-GA nanoparticles were adsorbed onto the surface of Se-MHA by electrostatic interaction to form Se-MHA / BG nanogels; Step 4: Use the nanogel described in step 3 to perform single-cell modification on the model probiotic EcN to prepare EcN@Se-MHA / BG.

[0021] Preparation, characterization, cellular uptake efficiency, and antioxidant and anti-inflammatory activities of BSA-GA nanoparticles, including: 1. Preparation Step 101: Dissolve serum albumin (BSA) in double-distilled water, stir for 2 hours, and store at 4° C. overnight to fully hydrate, to prepare a 50 μM bovine serum albumin (BSA) working solution. Step 102: slowly dropwise adding anhydrous ethanol solution containing different concentrations of galangin GA to the serum albumin BSA solution obtained in step 101, and stirring for 2 hours in a dark environment to achieve a final ethanol concentration in the solution of less than 2%; Step 103: Rotary evaporate the bovine serum albumin (BSA) solution obtained in step 102 at 35° C. for 10 minutes to remove ethanol, and replenish the lost volume with double-distilled water; Step 104: The bovine serum albumin (BSA) solution obtained in step 103 is adjusted to pH 12 using 2M NaOH, and 0-100 μM galangin (GA) is dissolved in double-distilled water at the same pH value to achieve a specific concentration. Similarly, the aqueous solution containing galangin (GA) is slowly added dropwise to the serum albumin (BSA) solution and stirred for 2 hours in the dark. Finally, the pH is accurately adjusted back to 7.4.

[0022] 2. Characterization (1) The particle size of the samples was measured by dynamic light scattering using a Zetasizer Nano ZS90, and the zeta potential was measured by electrophoretic light scattering. (2) The two freeze-dried nanoparticles were adhered to a conductive carbon tape and treated with gold spraying under high vacuum. The surface structure was observed using a scanning electron microscope (SEM) at 3 kV.

[0023] 3. Cellular uptake RAW 264.7 cells were seeded on coverslips in 24-well plates at a density of 5×105 cells per well and cultured at 37°C and 5% CO2 for 20 hours. Subsequently, the cells were cultured with two nanoparticles labeled with fluorescein isothiocyanate (FITC) (50 μM) for 4 hours, rinsed with PBS, and fixed with 4% paraformaldehyde for 20 minutes. The cells were then stained with a mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI) for 5 minutes. Finally, the cells were fixed with coverslips and fluorescence images were recorded using a confocal laser scanning microscope (CLSM), as shown in Figure 2. Figure 2 (A) Shown are BSA-GAd and BSA-GApH uptake in RAW264.7 cells after 4 hours of incubation at 37°C. Bright field (BF) confocal laser scanning microscopy images of DAPI-stained cell nuclei (blue), FITC-labeled nanoparticles (green), and their fusion layer. Images were processed using OLYMPUS image processing software.

[0024] 4. Cellular uptake rate RAW 264.7 cells were seeded on coverslips in 24-well plates at a density of 5 × 105 cells per well and cultured at 37°C, 5% CO2 for 20 hours. Subsequently, the cells were incubated with FITC-labeled nanoparticles for 0, 1, 2, and 4 hours. After completion, the cells were washed with PBS buffer, detached with 0.25% trypsin, and analyzed by flow cytometry. At least 10,000 events were recorded for each sample. The delivery efficiency of the nanoparticles was then studied by cellular uptake kinetics and fitted to the Michaelis-Menten equation:

[0025] Where Y represents the real-time uptake level, t represents the incubation time, and Y MAX represents the maximum uptake level at which the kinetic curve reaches saturation, and k is the uptake rate constant, representing the uptake time required to reach half of the maximum uptake level. Therefore, the smaller the k value, the faster the uptake rate of the nanoparticles. Figure 2 (B, C) Shown are the cellular uptake kinetics (B), the saturated cellular uptake value (Ymax), and the uptake rate constant k (C).

[0026] 5. Antioxidant activity The free radical scavenging activities of the two nanoparticles were investigated by DPPH and ABTS methods; Figure 3 Figure 4. Cellular antioxidant properties and inflammation-alleviating effects of BSA-GA nanoparticles and their components treated by desolvation and pH shift methods. ABTS and DPPH scavenging abilities (A), cytotoxicity against RAW264.7 cells (B), effects on ROS levels (C), LDH release (D), and secretion of pro-inflammatory cytokines (TNFα, IL-1β, IL-6) and anti-inflammatory cytokines (IL-10) (EH) in RAW264.7 cells treated with H2O2.

[0027] The ABTS working solution was prepared as follows: 7 mM ABTS solution was mixed with 2.45 mM potassium persulfate solution, allowed to stand in the dark for 16 hours, and diluted 20-fold before the assay. The DPPH working solution was prepared by dissolving DPPH powder in anhydrous ethanol to prepare a 0.1 mM solution, which was diluted 10-fold before use. For the ABTS assay, 50 μL of the nanoparticle solution and its equivalent components were added to 150 μL of the ABTS working solution, and incubated in the dark at 25°C for 7 minutes, followed by double-distilled water as a blank control. The absorbance of the sample was measured at 734 nm using a microplate reader. For the DPPH assay, the DPPH solution was thoroughly mixed with the nanoparticles and their components in equal volumes and incubated in the dark at 25°C for 30 minutes. The absorbance of the sample was measured at 517 nm using anhydrous ethanol as a control. The free radical scavenging ability was determined by the following formula:

[0028] Where A1 and A0 represent the absorbance of the sample group and the control group at specific wavelengths, respectively;

[0029] Anti-inflammatory activity: H2O2 was used to induce RAW264.7 cell damage, and the anti-inflammatory effect of the nanoparticles and their components was evaluated by analyzing the cell supernatant. The cytokines: tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-10 (IL-10) were measured according to the kit instructions. The specific operation is as follows: 100 μL of cell culture supernatant was added to the sample well, followed by the addition of antibodies, horseradish peroxidase-labeled streptavidin, and substrates. The absorbance at a wavelength of 450 nm was measured using a microplate reader to calculate the concentration of inflammatory factors; Figure 5 Shown are the scattered light intensity change curves of Se-MHA / BG and HA / BG at pH 7.4-1 (A) and 0-400μM H2O2 concentration (B).

[0030] Regarding the preparation of responsive Se-MHA / BG nanogels, including, Step 201, dissolving 68.5 mg of polybutylene adipate (PBA) and 90 mg of mannose in 20 mL of methanol, and reacting at room temperature for 6 hours to prepare an intermediate of mannose phenylboronic acid; Step 202, dissolving 200 mg of hyaluronic acid (HA) in 20 mL of deionized water, and slowly adding 155 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 86.3 mg of activated methoxypolyethylene glycol (NHS) to the solution, and stirring at room temperature for 60 minutes at pH 5 to activate the carboxyl groups on the hyaluronic acid (HA); Step 203, then continuing to add phenylboronic acid mannose to the solution and adjusting the solution to pH 8, stirring for 24 hours to obtain MHA. In order to remove unreacted molecules, the reaction product is dialyzed three times and collected by freeze drying; Step 204, completing the diselenide bridging of MHA, dissolving 200 mg of MHA in 20 mL of deionized water, and activating the carboxyl groups according to step 202, adding 159 mg of selenocystamine hydrochloride to the above mixture, reacting in the dark for 24 hours, dialyzing the reaction product three times, and lyophilizing to obtain Se-MHA powder; Se-MHA solution (5 mg / mL) and BSA-GA (BG) solution (5 mg / mL) were prepared separately and mixed thoroughly at a volume ratio of 4:1, so that the BG nanoparticles were adsorbed on the surface of the Se-MHA network by electrostatic interaction to form Se-MHA / BG nanogels. Figure 6 As shown, the formation diagram and microstructure of EcN@Se-MHA / BG (A), the particle size distribution and zeta potential difference of EcN and EcN@Se-MHA / BG (B and C), the pH response and H2O2 responsive phase behavior of EcN@Se-MHA / BG and EcN@HA / BG (D).

[0031] EcN was used as a model strain in this study. A single EcN colony was picked from a LB agar plate and inoculated into 5 mL of LB liquid medium at 37°C at 200 rpm overnight. After washing the EcN cells three times with 0.5 M NaCl, a 5 mg / mL Se-MHA / BG solution was added. The cells were stirred at 600 rpm with a magnetic stirrer for 2 hours and then centrifuged at 5000 g for 10 minutes to obtain EcN@Se-MHA / BG. The sample was dropped onto a copper grid and air-dried before being characterized by TEM.

[0032] 1. Growth curve: EcN, EcN@HA / BG, and EcN@Se-MHA / BG were inoculated into 50 mL of LB medium and cultured at 37°C with shaking to an initial OD600 of 0.17. OD values ​​were recorded every hour for 12 hours using a microplate reader to plot bacterial growth curves. Figure 7 Shown are the growth curves of EcN, EcN@HA / BG and EcN@Se-MHA / BG.

[0033] 2. In vitro simulated digestion experiment: Simulated gastric fluid (SGF) was prepared by adding 32 mg of pepsin to 100 mL of deionized water and adjusting the pH to 2.5; simulated intestinal fluid (SIF) was prepared by adding 1 g of trypsin and bile salt (4 mg / mL) to 100 mL of deionized water

[140] . Subsequently, equal amounts of EcN, EcN@HA / BG, and EcN@Se-MHA / BG (1×108 CFU / mL) were mixed with SGF at a ratio of 1:1 (v / v) and cultured at 37°C and 200 rpm for 2 hours, and then added to SIF at a ratio of 1:1 (v / v) and cultured for 4 hours. Samples at different digestion stages were observed by TEM. In order to determine the effect of the simulated digestion process on the activity of EcN, 500 μL of the sample was taken at each time point, washed with PBS, and inoculated into LB solid medium. The bacteria were counted after overnight culture; Figure 8 As shown, schematic diagram of the responsive phase behavior of EcN@Se-MHA / BG in simulated gastrointestinal digestion and mucus layer (A), the survival of probiotics of EcN, EcN@HA / BG and EcN@Se-MHA / BG in simulated gastrointestinal digestion (B), the micromorphological characteristics and bacterial plating in simulated gastrointestinal digestion (C).

[0034] 3. Motility and degradation analysis in simulated mucus layer: A porcine intestinal mucin solution supplemented with 200 μM H₂O₂ was used to simulate the inflamed intestinal mucus layer microenvironment. Subsequently, 10 μL of EcN or EcN@Se-MHA / BG (1×10⁻ CFU / mL) was added to 1 mL of the mucin solution. Bacterial movement was recorded over 20 seconds using an optical microscope with a video camera, and the total displacement distance and velocity were analyzed using Python-based software. To characterize the degradation of Se-MHA / BG in the mucus layer, Se-MHA / BG and EcN were stained with rhodamine B and Hoechst 33342, respectively, based on the aforementioned method. The mucin solution was stained with FITC-conjugated wheat germ agglutinin (FITC-WGA). CLSM tomography was used to record the state of Se-MHA / BG in the simulated mucus layer at the initial stage and 1 hour after addition, using excitation wavelengths of 352 nm, 488 nm, and 544 nm. In addition, the cleavage of diselenide bonds was analyzed by the H2O2 detection kit. After adding EcN@Se-MHA / BG to the porcine gastrointestinal mucin solution containing 200 μM H2O2, the changes in H2O2 levels were measured every 10 minutes; Figure 9Trajectory analysis (A), average moving velocity (B) and distance from the initial position (C) of EcN and EcN@Se-MHA / BG in simulated mucus layer, degradation (D) and H2O2 scavenging (E) of EcN@Se-MHA / BG in simulated mucus layer.

[0035] 4. Targeted adhesion analysis of colon inflammation sites: Se-MHA / BG nanocoatings for in vivo imaging were prepared by Cy5.5-labeled BG. C57BL / 6J mice were randomly divided into four groups: H2O + EcN@Se-MHA / BG-Cy5.5, H2O + EcN@HA / BG-Cy5.5, DSS + EcN@Se-MHA / BG-Cy5.5 and DSS + EcN@HA / BG-Cy5.5, n = 5 in each group. Mice were adapted for 7 days before the experiment, and then free drinking with 3% DSS or water for 6 days according to the grouping, and then gavage with EcN@Se-MHA / BG and EcN@HA / BG at a bacterial dose of 1 x 108 CFU according to the grouping on the 7th day, and the mice were anesthetized 6 hours after gavage, and the fluorescence in the mice was detected by IVIS. Then the mice were euthanized, and the colon was collected for further imaging. The total radiation efficiency of the tissue was analyzed using IVIS in vivo image analysis software. In addition, the colon tissue after imaging was fixed with 4% paraformaldehyde and paraffin-embedded, and continuous sectioning with a thickness of 10 μm. For immunofluorescence, the deparaffinized and rehydrated tissue was incubated with CD44 and CD206 primary antibodies and AexaFluor-488 and Cy3 cross-linking secondary antibodies to analyze the targeting of Cy5.5-labeled nanocoating to each receptor. After DAPI staining, the targeting of nanocoating to receptors was analyzed by CLSM at excitation wavelengths of 340, 488, 550 and 680 nm; as Figure 10 The establishment and administration of DSS-induced colitis mouse model for IVIS imaging (A), the fluorescence distribution images of EcN@HA / BG and EcN@Se-MHA / BG in healthy mice and colitis mice intestinal tract 6 hours after administration (B and C) and the total radiation efficiency in small intestine and colon (D and E) after Cy5.5 staining; as Figure 11 The co-localization fluorescence analysis of Se-MHA / BG and HA / BG with CD44 and CD206 receptors in inflammatory colon.

[0036] 5. Analysis of the colonization ability of probiotics: To determine the protection of Se-MHA / BG nanocoating on EcN and intestinal colonization. According to the above method, the DSS-induced colitis mouse model was established by free drinking 3% DSS for 6 days, and 1 x 108 CFU of EcN, EcN@HA / BG and EcN@Se-MHA / BG were used for single gavage on mice on the 7th day, respectively. Intestinal contents were collected at 6, 12, 24, 48 and 72 hours after gavage, respectively, diluted 1000 times with PBS, and cultured on eosin-methylene blue E. coli selective medium at 37°C overnight, and bacterial counting was performed; as shown in Figure 12 Figure 6, quantitative analysis of E. coli in intestinal contents of mice after gavage treatment of EcN, EcN@HA / BG and EcN@Se-MHA / BG (A) and representative image of eosin-methylene blue plate colonies after dilution (B).

[0037] 6, Therapeutic effect of EcN@Se-MHA / BG on DSS-induced colitis C57 mice were randomly divided into five groups: negative control (water + PBS), positive control (3% DSS + PBS), EcN (3% DSS + EcN), EcN@HA / BG (3% DSS + EcN@HA / BG) and EcN@Se-MHA / BG (3% DSS + EcN@Se-MHA / BG). As described above, C57BL / 6J mice were adapted to adaptive feeding for 7 days before the experiment, then 3% (w / v) DSS was added to the drinking water for 6 days to induce acute colitis, followed by gavage of 1 x 108 CFU of EcN, EcN@HA / BG and EcN@Se-MHA / BG on the 7th day to the 12th day, respectively, and the mice were euthanized after eyeball blood sampling on the 12th day, and the colon length and spleen weight of the mice were recorded after dissection. During the experiment, the body weight, visible stool consistency and fecal occult blood of the mice were recorded every day, and the corresponding disease activity index (DAI) was calculated. Scoring criteria: weight loss (<1%, 0; 1-5%, 1; 5-10%, 2; 10-18%, 3; >18%, 4), fecal occult blood (normal, 0; darker color, 1; slight bleeding, 2; bleeding, 3; severe bleeding, 4), stool consistency (normal, 0; basically formed, 1; partially and not formed, 2; very soft, 3; watery stool, 4); as shown in Figure 13As shown, DSS was induced, and mice were euthanized after eye blood was collected on the 12th day. The colon length and spleen weight of the mice were recorded after autopsy. The weight, visible stool consistency and fecal occult blood of the mice were recorded every day during the experiment, and the corresponding disease activity index (DAI) was calculated. Scoring criteria: weight loss (<1%, 0; 1-5%, 1; 5-10%, 2; 10-18%, 3; >18%, 4), fecal occult blood (normal, 0; darker color, 1; slight bleeding, 2; bleeding, 3; severe bleeding, 4), fecal consistency (normal, 0; basically formed, 1; partially and unformed, 2; very soft, 3; watery stool, 4); as shown Figure 13 Figure 2: Schematic diagram of DSS-induced colitis treatment in mice (A), changes in body weight and disease activity index (DAI) of mice in different treatment groups over 12 days (B and C), images of colon and spleen tissues in different prevention groups (D), colon length, and spleen index (E and F).

[0038] 7. EcN@Se-MHA / BG protects the intestinal epithelial barrier: Colonic tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for H&E, AB-PAS, and TUNEL staining. The degree of colonic injury was assessed using the histochemical colitis scoring system: tissue inflammation: none, 0; mild, 1; moderate, 2; severe, 3. Lesion depth: none, 0; mucosal, 1; submucosa, 2; transmural, 3. Crypt destruction: none, 0; basal 1 / 3 damage, 1; basal 2 / 3 damage, 2; crypt loss with presence of surface epithelium, 3; and crypt and surface epithelium loss, 4. Ulcer area: 0%, 0; 1%-25%, 1; 26%-50%, 2; 51%-75%, 3; and 76%-100%, 4. Subsequently, the expression of tight junction proteins (ZO-1 and occludin) and colonic mucin was semiquantitatively analyzed by immunofluorescence. According to the above method, colon sections were dewaxed and rehydrated, and then ZO-1 and occludin primary antibodies were added, respectively. After incubation at 4°C overnight, fluorescently labeled secondary antibodies were added and incubated at room temperature for 1 hour. Finally, DAPI staining was used for 10 minutes at room temperature in the dark. After washing with PBS, the colon tissue was photographed by CLSM at 340 nm, 488 nm, and 594 nm. Mucin was labeled with FITC-WGA probe for 1 hour and stained with DAPI staining for 10 minutes at room temperature in the dark. After washing with PBS, the colon tissue was photographed at 340 nm and 488 nm. All fluorescence images were analyzed for relative fluorescence intensity using ImageJ software; Figure 14Shown are hematoxylin & eosin (H&E) staining, AB-PAS staining, TUNEL staining images (A) and histochemical scores (B) of intestinal tissue sections of mice in different treatment groups, and immunofluorescence staining images (C) and semi-quantitative analysis (D) of mucin and tight junction proteins (ZO-1 and Occludin) in intestinal tissues of mice in different treatment groups.

[0039] 8. Analysis of intestinal macrophage polarization level: After dewaxing and rehydration, the colon sections were treated with CD86 primary antibody and AexaFluor-488 cross-linked secondary antibody to label M1 macrophages, while M2 macrophages were labeled with CD206 primary antibody and AexaFluor-594 cross-linked secondary antibody. Finally, DAPI staining solution was used for 10 minutes at room temperature in the dark. The changes in the expression levels of M1 / M2 macrophages in colon tissue were recorded by CLSM at excitation wavelengths of 340 nm, 488 nm, and 594 nm, and the relative fluorescence intensity was calculated by ImageJ software. Figure 15 Shown are the immunofluorescence images of CD86 and CD206 receptors in colon tissue sections of different treatment groups (A) and the changes in the ratio of M2 to M1 macrophages (B).

[0040] 9. Determination of related inflammatory factors and antioxidant enzymes: The collected mouse blood samples were allowed to stand for 30 minutes and then centrifuged at 2000 g for 10 minutes at 4°C to separate the serum. The corresponding inflammatory factors, TNF-α, IL-6, IL-1β and IL-10, were determined according to the instructions of the kit. The level of intestinal oxidative stress was assessed by the activity of relevant antioxidant enzymes: myeloperoxidase (MPO), SOD and GSH-px, as well as the content of the oxidation marker malondialdehyde (MDA). The collected colon tissue was added to the preparation solution in the kit and homogenized in an ice bath. Subsequently, the supernatant was centrifuged at 12000 g for 10 minutes at 4°C and tested according to the instructions provided by the kit; Figure 16 Shown are the levels of inflammatory markers TNF-α (A), IL-6 (B), IL-1β (C), and IL-10 (D), the level of oxidative stress marker MDA (E), and the activities of related antioxidant enzymes MPO, GSH-px, and SOD (FH) in colon tissue.

[0041] 10. Intestinal microbial and metabolomics analysis: After one week of adaptive feeding, in addition to the normally fed healthy group, the model group was treated with 3% DSS for 6 days to induce acute colitis, and then 200 μL of 1×108 CFU EcN@Se-MHA / BG was gavaged daily for 6 days. Conversely, the prevention group was gavaged with 200 μL of 1×108 CFU EcN@Se-MHA / BG daily for the first 6 days, followed by 3% DSS treatment for 6 days to induce acute colitis. Uncoated EcN, EcN@HA / BG, and PBS were used as controls. Feces of mice in each group were then collected for subsequent microbiome and metabolome analyses, as shown in Figure 2. Figure 17 As shown, schematic diagram of the prevention of DSS-induced colitis in mice (A), changes in body weight and disease activity index (DAI) of mice in different treatment groups within 12 days (B and C), images of colon tissue and spleen tissue (D), colon length and spleen index (E and F) of different treatment groups; Figure 18 As shown, hematoxylin & eosin (H&E) staining, AB-PAS staining, TUNEL staining and histochemical scoring of intestinal tissue sections of mice in different treatment groups (A and B), CD86 and CD206 receptor immunofluorescence staining images and changes in the proportion of M2 and M1 macrophage phenotypes (C and D); Figure 19 As shown in Figure 2, the α-diversity (Shannon index, Simpson index, and Chao1 index) of the intestinal microbiota of mice in the DSS-induced colitis treatment experiment (AC) and prevention experiment (DF) were compared. Figure 20 As shown, the relative abundance of the intestinal microbiota at the phylum level and the relative abundance of Proteobacteria in the mouse in the DSS-induced colitis treatment experiment (A and B) and prevention experiment (C and D); Figure 21 As shown, the relative contents of some differential metabolites that were significantly changed in the DSS-induced colitis treatment experiment (AD) and prevention experiment (EH) in mice.

[0042] The present application discloses a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness, which is obtained by the preparation method. The nanogel has specific binding and responsiveness to the site of colon inflammation, promotes the colonization of EcN in the colon, effectively alleviates IBD symptoms, increases the diversity of microbial composition in the inflamed intestine, and regulates the ratio of Bacteroidetes to Firmicutes. The abundance of beneficial bacteria Lactobacillus is increased, and the abundance of pathogenic bacteria such as Bacteroides is reduced, which helps to increase the levels of various metabolites such as uridine and 2'-deoxyuridine in the intestinal purine metabolism and pyrimidine metabolism pathways, thereby improving inflammation.

[0043] The present invention relates to an application of a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness. The above-mentioned hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness is used to modify single cells of model probiotics to prepare nanogel EcN@Se-MHA / BG loaded with model probiotic Escherichia coli Nissle 1917 (EcN) as a carrier for oral probiotics.

[0044] The above description is only an implementation method of the present application and does not limit the patent scope of the present application. Any equivalent structure or equivalent process transformation made using the contents of the description and drawings of this application, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present application.

Claims

1. A method for preparing a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness, characterized in that: include: Galangin (GA) was loaded into bovine serum albumin (BSA) to prepare BSA-GA nanoparticles with antioxidant and anti-inflammatory activities. A multifunctional molecule, Se-MHA, was synthesized based on hyaluronic acid (HA) to target M2 macrophages and respond to ROS degradation. Se-MHA / BG nanogels were prepared by mixing BSA-GA nanoparticles (BG) as anti-inflammatory functional components with Se-MHA at a volume ratio of 4:

1. Se-MHA / BG nanogel was prepared and single-cell modification of the model probiotic EcN was performed to prepare the probiotic-loaded nanogel EcN@Se-MHA / BG.

2. The method for preparing a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness according to claim 1, characterized in that: The method comprises: Step 1, BSA-GA nanoparticles (BG) were prepared by loading the hydrophobic flavonoid galangin (GA) with anti-inflammatory activity into bovine serum albumin (BSA) via ethanol desolvation and pH shift methods respectively; Step 2: MHA is prepared by modifying mannose groups on hyaluronic acid (HA) molecules, and further cross-linking them using diselenide bonds to form a continuous network Se-MHA; Step 3: The BSA-GA nanoparticles obtained in step 1 are adsorbed onto the surface of Se-MHA by electrostatic interaction to form Se-MHA / BG nanogels; Step 4: Use the nanogel described in step 3 to perform single-cell modification on the model probiotic EcN to prepare EcN@Se-MHA / BG.

3. The method for preparing a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness according to claim 2, characterized in that: The ethanol desolvation method comprises: Step 11, slowly adding an anhydrous ethanol solution containing 0-100 μM galangin GA to a bovine serum albumin (BSA) solution and stirring for 2 hours in the dark, with the final ethanol concentration controlled within 2%; In step 12, ethanol was removed by rotary evaporation at 35°C for 10 minutes, and the lost volume was replenished with double-distilled water.

4. The method for preparing a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness according to claim 2, characterized in that: The pH shift method comprises: Step 21, adjust the pH to 12 with 50 μM bovine serum albumin BSA. Step 22: slowly add 0-100 μM galangin GA solution dropwise into bovine serum albumin BSA, stir for 2 h in the dark, and finally adjust the pH to 7.4 to obtain BSA-GA nanoparticles BG.

5. The method for preparing a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness according to any one of claims 1 to 4, characterized in that: The method for preparing the BSA-GA nanoparticles comprises: Step 101, dissolving bovine serum albumin (BSA) in double-distilled water, stirring for 2 hours, and storing at 4° C. overnight to fully hydrate, to prepare a 50 μM bovine serum albumin (BSA) working solution; Step 102: slowly dropwise adding a 0-100 μM galangin GA solution in anhydrous ethanol to the bovine serum albumin (BSA) solution obtained in step 101, and stirring for 2 hours in a dark environment to achieve a final ethanol concentration in the solution of less than 2%; Step 103: Rotary evaporate the bovine serum albumin (BSA) solution obtained in step 102 at 35° C. for 10 minutes to remove ethanol, and replenish the lost volume with double-distilled water; Step 104: The bovine serum albumin (BSA) solution obtained in step 103 is adjusted to pH 12 using 2M NaOH, and 0-100 μM galangin (GA) is dissolved in double-distilled water at the same pH value to achieve a final concentration controlled within 2%. Similarly, the aqueous solution containing galangin (GA) is slowly added dropwise to the bovine serum albumin (BSA) solution and stirred for 2 hours in the dark. Finally, the pH value is accurately adjusted back to 7.

4.

6. The method for preparing a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness according to any one of claims 1 to 4, characterized in that: The preparation method of the Se-MHA / BG nanogel comprises: Step 201, dissolving 68.5 mg of polybutylene adipate (PBA) and 90 mg of mannose in 20 mL of methanol, and reacting at room temperature for 6 hours to prepare an intermediate of mannose phenylboronic acid; Step 202, dissolving 200 mg of hyaluronic acid (HA) in 20 mL of deionized water, and slowly adding 155 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and 86.3 mg of activated methoxypolyethylene glycol (NHS) to the solution, and stirring at room temperature for 60 minutes at pH 5 to activate the carboxyl groups on the hyaluronic acid (HA); Step 203, then continuing to add phenylboronic acid mannose to the solution and adjusting the solution to pH 8, stirring for 24 hours to obtain MHA. In order to remove unreacted molecules, the reaction product is dialyzed three times and collected by freeze drying; Step 204, completing the diselenide bridging of MHA, dissolving 200 mg of MHA in 20 mL of deionized water, and activating the carboxyl groups according to step 202, adding 159 mg of selenocystamine hydrochloride to the above mixture, reacting in the dark for 24 hours, dialyzing the reaction product three times, and lyophilizing to obtain Se-MHA powder; 5 mg / mL Se-MHA solution and 5 mg / mL BSA-GA (BG) solution were prepared separately and thoroughly mixed at a volume ratio of 4:1, so that the nanoparticles BG were adsorbed on the surface of the Se-MHA network through electrostatic action to form Se-MHA / BG nanogel.

7. A hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness, characterized in that: The product is obtained by the preparation method according to any one of claims 1 to 6.

8. Application of a hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness, characterized in that: The model probiotics were modified with the hyaluronic acid-based nanogel with intestinal targeting and inflammatory responsiveness as described in claim 7 to prepare the nanogel EcN@Se-MHA / BG loaded with the model probiotic EcN as a carrier for oral probiotics.

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