ROS-sensitive hydrogel as well as preparation method and application thereof

Abstract

The invention discloses ROS (reactive oxygen species) sensitive hydrogel as well as a preparation method and application thereof. The ROS sensitive hydrogel disclosed by the invention is prepared from aminophenylboronic acid, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, hyaluronic acid, tannic acid and water. On the basis of ROS-sensitive dynamic boric acid ester bonds, aminophenylboronic acid modified HA (HA-PB) and tannic acid are mixed to prepare the hydrogel (HT). And adding a PVA (Polyvinyl Alcohol) solution into the HT hydrogel to obtain the hydrogel HTP. In the preparation process of the HTP hydrogel, the L.reuterii is added, so that the L.reuterii coated HTP hydrogel can be obtained. The hydrogel disclosed by the invention has excellent biological safety and degradability, the hydrogel can efficiently release TA to relieve an oxidative stress reaction due to ROS responsive degradation at an inflammation part, and HA released during degradation is also beneficial to repair of damaged gastric mucosa. More importantly, the homeostasis of intestinal flora is not influenced by using the therapy.

Description

Technical Field

[0001] The invention relates to the field of biomaterials, and in particular to a ROS-sensitive hydrogel loaded with L. reuteri, a preparation method thereof, and an application thereof in treating gastritis caused by Helicobacter pylori infection. Background Art

[0002] Helicobacter pylori (HP), as a microorganism that parasitizes the stomach, has always been the focus of attention in the study of gastric diseases. About half of the world's population is affected by it. Studies have shown that HP is one of the main factors causing gastritis. Its spiral shape enables it to shuttle through the mucus layer in the stomach, survive in a high-concentration gastric acid environment, and parasitize on the surface of the gastric mucosa. This parasitism triggers the body's immune response, thereby causing chronic gastric mucosal inflammation. Under oxidative stress, the gastric mucosa is further damaged, and cell membrane lipid peroxidation destroys the integrity and permeability of the cell membrane. In addition, oxidative stress also affects the adhesion molecules of gastric mucosal epithelial cells, exacerbating the adhesion and infiltration of inflammatory cells. At present, the treatment for HP infection is mainly triple or quadruple drug therapy containing two antibiotics. However, with the continuous increase in antibiotic resistance, traditional treatment methods have problems such as a continuous decline in the success rate of positive treatment, significant side effects, and a single efficacy. Summary of the invention

[0003] The present invention provides a ROS-sensitive hydrogel loaded with Lactobacillus reuteri (L. reuteri) for treating gastritis caused by HP infection, which can solve one or more of the above-mentioned problems in the prior art.

[0004] According to one aspect of the present invention, a ROS-sensitive hydrogel is provided, comprising 0.5-1.5 g of aminophenylboronic acid (HB), 0.1-1 g of N-hydroxysuccinimide (NHS), 0.1-1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), 0.5-2 g of hyaluronic acid (HA) and 0.5-2 g of tannic acid (TA), and the rest is water.

[0005] The beneficial effect of the present invention is that based on the ROS-sensitive dynamic boronate bond, aminophenylboronic acid-modified HA (HA-PB) is mixed with tannic acid to prepare a hydrogel (HT). The hydrogel of the present invention has excellent biosafety and degradability. The ROS-responsive degradation at the inflammatory site enables it to efficiently release TA to alleviate oxidative stress, and the HA released during degradation also helps to repair damaged gastric mucosa. More importantly, the use of this therapy will not affect the homeostasis of intestinal flora.

[0006] Hyaluronic acid is a polysaccharide compound widely found in connective tissue, joint fluid and vitreous body of the eye, and has excellent biocompatibility. HA can regulate the activity of immune cells and inhibit the occurrence of inflammatory response by interacting with cell surface receptors; in addition, HA can also interact with the intercellular matrix, promote cell migration and proliferation, and accelerate the repair of damaged tissues.

[0007] Tannic acid is a natural compound mainly found in plants. It uses the ability of phenolic hydroxyl groups in its molecular structure to capture free radicals, greatly reducing cell damage caused by oxidative stress. Therefore, the hydrogel prepared based on TA has anti-inflammatory effects.

[0008] In some embodiments, 0.5-1 g of polyvinyl alcohol (PVA) is also included. The beneficial effect is that biocompatible macromolecular polyvinyl alcohol is added to the hydrogel to further improve the mechanical properties of the hydrogel by utilizing hydrogen bonds.

[0009] In some embodiments, Lactobacillus reuteri is also included. The beneficial effect is that the loaded L. reuteri can treat HP infection, and L. reuteri can affect the survival and colonization of HP, thereby playing a positive role in gastrointestinal health.

[0010] According to another aspect of the present invention, a method for preparing a ROS-sensitive hydrogel is provided, characterized in that it comprises:

[0011] A. Dissolve 0.5-2 g of HA in 100 mL of deionized water, add 0.5-1.5 g of aminophenylboronic acid, 0.1-1 g of N-hydroxysuccinimide and 0.1-1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir evenly at room temperature to obtain a yellow viscous solution; dialyze the viscous solution in deionized water to remove unreacted aminophenylboronic acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; after the dialysis, freeze-dry the product to obtain HA-PB;

[0012] B. Evenly mix the HA-PB aqueous solution and the tannic acid aqueous solution and stir to obtain a hydrogel.

[0013] The beneficial effect is that the carboxyl groups on HA are activated to form active esters through the "EDC / NHS" carbodiimide coupling method, and then reacted with 3-aminophenylboronic acid to produce HA-PB. Based on the ROS responsiveness of the dynamic boronate ester bond, HA-PB and TA are bonded through the boronate ester bond to form a hydrogel with ROS responsiveness.

[0014] In some embodiments, in step B, the w / v concentration of the HA-PB aqueous solution is 4%, the w / v concentration of tannic acid is 4%, and the volume ratio of the HA-PB solution to the tannic acid solution is 3: 1. The beneficial effect is that the appropriate component ratio can prepare an injectable hydrogel with suitable mechanical properties.

[0015] In some embodiments, it is characterized in that step B further comprises adding polyvinyl alcohol to the hydrogel. The beneficial effect is that the addition of PVA introduces hydrogen bonds, increases the cross-linking density of the hydrogel and thus slows down the degradation rate, thereby producing HTP hydrogel.

[0016] In some embodiments, it also includes dissolving polyvinyl alcohol in water, the w / v concentration of polyvinyl alcohol is 2%, and the volume ratio of HA-PB solution to polyvinyl alcohol solution is 3:(0.5-2). The beneficial effect is that TA, as a large molecular weight water-soluble compound, exhibits significant antioxidant properties due to its multiple phenolic hydroxyl groups. PVA, as a water-soluble polymer, contains multiple hydroxyl groups, which give it good hydrophilicity and the ability to form hydrogen bonds. In this study, the phenylboronic acid group of HA-PB and the ortho-diphenol group of TA react rapidly to form a borate ester bond to prepare HT hydrogel, and then PVA is added thereto to obtain HTP hydrogel.

[0017] In some embodiments, step B further comprises adding L. reuteri to the HA-PB solution, wherein the amount of L. reuteri is (0.5-2)×10 8 CFU / mg. The beneficial effect is that the L. reuteri-loaded hydrogel L. reuteri@HTP has excellent antibacterial ability.

[0018] According to another aspect of the present invention, one or more applications of the hydrogel in removing ROS, inhibiting the activity of HP, repairing gastric mucosa and stabilizing intestinal flora are provided.

[0019] Its beneficial effect is that TA is an efficient ROS scavenger and can effectively alleviate the oxidative stress response of tissues. The scavenging ability of nitrogen-centered free radicals and oxygen-centered free radicals of HTP hydrogel was tested using DPPH· and PTIO· free radical scavenging assays. HTP hydrogel is rich in HA, which can promote adhesion molecules such as integrins that affect the cell surface, regulate the adhesion and detachment of cells from the extracellular matrix, and can also interact with receptors on the cell surface (such as CD44), activate internal signal transduction pathways, and promote cell proliferation and differentiation. Therefore, L. reuteri@HTP hydrogel has great potential in promoting gastric mucosal repair. L. reuteri@HTP hydrogel can effectively alleviate gastric inflammation in mice while eradicating HP in the stomach. As a multifunctional polysaccharide, HA may promote the colonization and proliferation of L. reuteri in the intestine by maintaining water balance and providing extracellular matrix. In addition, the biocompatibility and immunomodulatory properties of HA provide a more favorable intestinal microenvironment for L. reuteri, thereby optimizing the composition and function of the microbial community.

[0020] The L.reuteri@HTP hydrogel of the present invention is based on the dynamic boronate bond of TA and HA-PB. This hydrogel can responsively release L.reuteri, and the degradation rate of the hydrogel in vivo is controlled by adding PVA thereto. Both in vivo and in vitro experiments confirm that this hydrogel can effectively inhibit the activity of HP; cell scratches and immunofluorescence staining show that the migration rate of cells treated with L.reuteri@HTP is increased, the expression of β-catenin and PCAN is increased, and the damaged gastric mucosa is restored; HP mice are scored for gastritis, and the degree of inflammation in the treated mice is significantly reduced, and the expression of related inflammatory factors is decreased. Analysis of the intestinal flora of HP mice after treatment shows that the flora abundance of the experimental group mice is similar to that of healthy mice, confirming that L.reuteri@HTP hydrogel does not affect the homeostasis of intestinal flora. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 a is the synthetic route of HA-PB;

[0022] Figure 1 b is the FTIR curves of HA and HA-PB;

[0023] Figure 1 c is the 1H NMR curves of HA and HA-PB;

[0024] Figure 2 a is a digital photo of HA-PB, HTP and L. reuteri@HTP;

[0025] Figure 2b is the SEM images of HT, HTP and L. reuteri@HTP;

[0026] Figure 2 c is a partial magnified SEM image of HT, HTP and L. reuteri@HTP (L. reuteri is marked in green);

[0027] Figure 3 a is the SEM image of L. reuteri;

[0028] Figure 3 b is the injectability of HTP hydrogel;

[0029] Figure 4 a is a digital photo of HT and HTP hydrogel degradation under different environments;

[0030] Figure 4 b is the degradation curve of HT hydrogel;

[0031] Figure 4 c is the degradation curve of HTP hydrogel;

[0032] Figure 5 a shows the survival of L. reuteri under confocal microscopy (green mark);

[0033] Figure 5 b is the enlarged image of the survival of L. reuteri (green mark) under confocal microscope;

[0034] Figure 5 c shows the survival of L. reuteri after 2 h of incubation in SGF;

[0035] Figure 6 a is the SEM image of HP and Lactobacillus reuteri after incubation in different environments;

[0036] Figure 6 b shows the growth of HP on blood agar plates after incubation with different groups;

[0037] Figure 6 c shows the survival of HP after SYTO 9 / PI live / dead bacteria double staining;

[0038] Figure 7 a is the DPPH·scavenging rate of HTP hydrogel and the digital photo of the scavenging process;

[0039] Figure 7 b shows the PTIO· removal rate of HTP hydrogel and the digital photos of the removal process;

[0040] Figure 7chFlow cytometry was used to determine the intracellular ROS scavenging ability of HTP hydrogel;

[0041] Figure 8 a is the live-dead staining image of L. reuteri@HTP hydrogel extracts and gastric epithelial mucosal cells at 24 h with concentrations of 5, 10, 15 and 20 mg / mL;

[0042] Figure 8 b is the live-dead staining images of L. reuteri@HTP hydrogel extracts and gastric epithelial mucosal cells at 48 h at concentrations of 5, 10, 15, and 20 mg / mL;

[0043] Figure 8 c is the live-dead staining image of L. reuteri@HTP hydrogel extracts and gastric epithelial mucosal cells at 72 h with concentrations of 5, 10, 15, and 20 mg / mL, respectively;

[0044] Figure 8 d is the cell viability of L. reuteri@HTP hydrogel extracts and gastric epithelial mucosal cells at 72 h at concentrations of 5, 10, 15 and 20 mg / mL;

[0045] Figure 8 e is a digital photo of L. reuteri@HTP hydrogel co-incubated with red blood cells

[0046] Figure 8 f is the hemolysis rate of L. reuteri@HTP hydrogel co-incubated with red blood cells;

[0047] Fig. 9 a is the H&E stained sections of the main organs of the mice after treatment in different experimental groups: heart, liver, spleen, lung, and kidney;

[0048] Fig. 9 b to Fig. 9 i is the blood routine and blood biochemical indicators of mice after L. reuteri@HTP hydrogel treatment;

[0049] Fig.10 a is the images of cell migration after treatment in different experimental groups taken at 0h, 12h, 24h and 36h;

[0050] Fig.10 b is the migration rate of cells at 12h, 24h, and 36h;

[0051] Fig.11 a is the IVIS imaging photos of healthy mice and HP-infected mice treated with L. reuteri@HTP hydrogel for 2 hours;

[0052] Fig.11 b shows the fluorescence intensity of healthy mice and HP-infected mice after being treated with L. reuteri@HTP hydrogel for 2 hours;

[0053] Fig.12 a is a digital photo of mice infected with HP using rapid urease assay;

[0054] Fig.12 b is a digital photo of HP mouse blood agar plate culture; Fig.12 c is the H&E staining result of HP mouse stomach;

[0055] Fig.13 Immunofluorescence images of gastric mucosal repair in HP mice after treatment in different experimental groups;

[0056] Fig.14 a H&E staining of the stomach of HP mice after treatment in different experimental groups;

[0057] Fig.14 b to Fig.14 j is the immunohistochemistry of HP mice after treatment in different experimental groups;

[0058] Fig.15 a is the microbial community diversity index Chao1;

[0059] Fig.15 b is the Shannon index of microbial community diversity;

[0060] Fig.15 c is the Simpson index of microbial community diversity;

[0061] Fig.15 d Weighted UniFrac distance matrix heat map showing the differences in microbial community composition among treatment groups;

[0062] Fig.15 e Principal coordinate analysis based on weighted UniFrac distance;

[0063] Fig.15 f Non-metric multidimensional scaling (NMDS) analysis;

[0064] Fig.15 g Distribution diagram of relative abundance of bacteria in each treatment group.

[0065] Fig.15 h Relative abundance distribution of Lactobacillus in each treatment group. DETAILED DESCRIPTION

[0066] The present invention will be further described in detail below in conjunction with the accompanying drawings.

[0067] Example 1

[0068] 1g of HA was fully dissolved in 100mL of deionized water, and 0.86g of aminophenylboronic acid, 0.58g of N-hydroxysuccinimide (NHS) and 0.96g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added thereto, and stirred at room temperature for 48h to obtain a yellow viscous solution. The solution was placed in a dialysis bag with a molecular weight cutoff of 3000MDa and dialyzed in deionized water for 5 days to remove unreacted PBA and EDC / NHS. After the dialysis, the product was placed in a -20℃ refrigerator overnight and freeze-dried to obtain HA-PB. In order to verify the successful preparation of HA-PB, the structural information of HA and HA-PB in the range of 4000-400cm-1 was observed and recorded by Fourier transform infrared spectrometer (FTIR). The structural characteristics of HA and HA-PB were further analyzed by 1H NMR.

[0069] like Figure 1 As shown in a, the carboxyl group on HA is activated to form an active ester through the classic "EDC / NHS" carbodiimide coupling method, and then reacted with 3-aminophenylboronic acid to obtain HA-PB. Figure 1 b is the FTIR spectra of HA and HA-PB. In the figure, the new stretching peaks formed by HA-PB at 1636, 1554 and 1482 cm-1 are due to the bending of the C=C double bond of the benzene ring. In addition, new HA-PB absorption peaks can be observed at 1340 and 705 cm-1, which are related to the stretching vibration and CH bending vibration of the benzene ring, respectively. Figure 1 As shown in the 1H NMR spectrum of c, HA-PB has multiple peaks caused by the proton conjugate absorption on the benzene ring in phenylboronic acid at δ = 7.2-7.6ppm, while the absorption peaks at δ = 1.8-2.0ppm and δ = 4.78ppm are the methyl proton peak of HA and the solvent peak of D2O, respectively. The above experiments fully confirmed that 3-aminophenylboronic acid was successfully grafted onto HA. In order to understand the grafting rate of 3-aminophenylboronic acid in HA-PB, the integral of the proton conjugate absorption peak (7.2-7.6ppm) on the benzene ring in the phenylboronic acid group was compared with the integral of the methyl proton peak of HA, and the grafting rate of 3-aminophenylboronic acid in HA-PB was calculated to be 24.3%.

[0070] Example 2

[0071] 0.5 g of HA was fully dissolved in 100 mL of deionized water, and 0.5 g of aminophenylboronic acid, 0.1 g of N-hydroxysuccinimide (NHS) and 0.1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added thereto, and stirred at room temperature for 48 h to obtain a yellow viscous solution. The solution was placed in a dialysis bag with a molecular weight cutoff of 3000 MDa and dialyzed in deionized water for 3 days to remove unreacted PBA and EDC / NHS. After the dialysis, the product was placed in a -20 ° C refrigerator overnight and freeze-dried to obtain HA-PB.

[0072] Example 3

[0073] 2g of HA was fully dissolved in 100mL of deionized water, and 0.86g of aminophenylboronic acid, 0.58g of N-hydroxysuccinimide (NHS) and 0.96g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added thereto, and stirred at room temperature for 48h to obtain a yellow viscous solution. The solution was placed in a dialysis bag with a molecular weight cutoff of 3000MDa and dialyzed in deionized water for 5 days to remove unreacted PBA and EDC / NHS. After the dialysis, the product was placed in a -20°C refrigerator overnight and freeze-dried to obtain HA-PB.

[0074] Example 4

[0075] 2g of HA was fully dissolved in 100mL of deionized water, and 1.5g of aminophenylboronic acid, 1g of N-hydroxysuccinimide (NHS) and 1g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added thereto, and stirred at room temperature for 48h to obtain a yellow viscous solution. The solution was placed in a dialysis bag with a molecular weight cutoff of 3000MDa and dialyzed in deionized water for 5 days to remove unreacted PBA and EDC / NHS. After the dialysis, the product was placed in a -20°C refrigerator overnight and freeze-dried to obtain HA-PB.

[0076] Example 5

[0077] The freeze-dried HA-PB in Example 1 was dissolved in deionized water to prepare a HA-PB (4%, w / v) solution. TA (4%, w / v) and PVA (2%, w / v) solutions were prepared separately. 300 μL of HA-PB and 100 μL of TA were mixed and stirred to obtain a hydrogel HT. 100 μL of PVA solution was added to the HT hydrogel to obtain a hydrogel HTP. During the preparation of the HTP hydrogel, L. reuteri (1×10 8 CFU / mg) can be obtained by L. reuteri@HTP hydrogel.

[0078] HA-PB and TA were mixed evenly in the sample tube according to the recipe, and then the formation of hydrogel was confirmed by the vial inversion method ( Figure 2 a). HA-PB appears as a viscous milky white liquid in the sample tube, and can flow slowly along the tube wall after inversion; with the addition of TA, the substance in the sample tube quickly gels, showing the formation of a light yellow HT hydrogel, and no flow occurs when the sample tube is inverted. PVA solution is further added to the HT hydrogel and mixed evenly, and the formed HTP hydrogel is retained in the sample tube in a fixed form.

[0079] The freeze-dried samples of hydrogels with different compositions were observed by SEM. Figure 2 As shown in Figure b, it can be found that all hydrogel samples have a loose and porous sponge-like structure. The HTP hydrogel forms more abundant hydrogen bonds due to the addition of PVA, which is denser than the HT hydrogel, but has no obvious difference from the L. reuteri@HTP hydrogel.

[0080] To verify the successful loading of L. reuteri, SEM was used to observe the growth of L. reuteri and L. reuteri@HTP hydrogel ( Figure 2 c).

[0081] like Figure 3 As shown in a, L. reuteri showed a short rod-like morphology with obvious characteristics in SEM, most of which existed alone or in a small number of clusters. Figure 2 In c, after magnifying the L. reuteri@HTP hydrogel, a large number of L. reuteri-characteristic bacilli can be observed on the hydrogel surface. The surface of the HT hydrogel at the same magnification is relatively smooth, so we believe that the L. reuteri@HTP hydrogel was successfully prepared. It is worth mentioning that the HTP hydrogel exhibits excellent injectability, which is attributed to the breaking and reconstruction of the dynamic boronate ester bond ( Figure 3 b).

[0082] Example 6

[0083] Dissolve the freeze-dried HA-PB in Example 1 with deionized water to prepare HA-PB (4%, w / v) solution. In addition, prepare TA (4%, w / v) and PVA (2%, w / v) solutions respectively. Mix 300 μL of HA-PB and 100 μL of TA evenly and stir, and the prepared hydrogel is named HT. Add 50 μL of PVA solution to the HT hydrogel, and the obtained hydrogel is named HTP. Adding L. reuteri (1×108 CFU / mg) during the preparation of HTP hydrogel can obtain L. reuteri@HTP hydrogel.

[0084] Example 7

[0085] Dissolve the freeze-dried HA-PB in Example 1 with deionized water to prepare HA-PB (4%, w / v) solution. In addition, prepare TA (4%, w / v) and PVA (2%, w / v) solutions respectively. Mix 300μL of HA-PB and 100μL of TA evenly and stir, and the prepared hydrogel is named HT. Add 200μL of PVA solution to the HT hydrogel, and the obtained hydrogel is named HTP. By adding L.reuteri (1×108CFU / mg) during the preparation of HTP hydrogel, L.reuteri@HTP hydrogel can be obtained.

[0086] Example 8

[0087] In order to test the degradation ability of L.reuteri@HTP hydrogel under different environments. 0.15g of the freeze-dried L.reuteri@HTP hydrogel prepared in Example 5 was immersed in 10mL of simulated gastric fluid (SGF), simulated gastric fluid containing H2O2 (0.1% w / v, H2O2+SGF) and simulated intestinal fluid (SIF). The mixed material was incubated with shaking at 37°C (100rpm / min), and the container was taken out and photographed at 30min, 120min and 720min, respectively. The hydrogel was freeze-dried and the mass was recorded. The degradation rate of the hydrogel was calculated using the following formula:

[0088]

[0089] The hydrogel plays an important role in the treatment of gastritis caused by HP infection. Its suitable degradation rate in the stomach can ensure the responsive release of L. reuteri. Based on the ROS sensitivity of the dynamic boronate ester bond, it can be used in the treatment of gastritis caused by HP infection. - ) and other environments, thus promoting the rapid degradation of the hydrogel. Figure 4As shown in Figures ac, after immersion in gastric juice for 0.5h, the HT hydrogel rapidly disintegrated in SGF, with a degradation rate of 62.8±8.2%, 83.4±4.3% after 3h, and 90.8±3.0% in H2O2+SGF. The above results show that HT degrades rapidly in gastric juice, so that it is not significantly affected by ROS and cannot achieve the effect of ROS response well. Correspondingly, HTP hydrogel degraded by 68.7±3.4% in H2O2+SGF within 3h, while only 25.0±4.9% in SGF. This is mainly because hydrogen bonds give HTP hydrogel a higher cross-linking density, thereby delaying the excessive degradation of the hydrogel and achieving the purpose of controllable responsive degradation. It is worth noting that both HT and HTP hydrogels can be slowly degraded in SIF and completely dissipated after 12h. During the degradation process, due to the rupture of the borate ester bond, the TA inside the hydrogel is released and rapidly oxidized in the alkaline SIF, so that the color of the solution changes to brown-yellow. This suggests that even if the hydrogel is not digested in the stomach, it can eventually be rapidly degraded in the intestine.

[0090] Example 9

[0091] The viscoelasticity of the hydrogel was characterized in detail on a rotational rheometer using rheological methods. Among them, the storage modulus (G') reflects the elastic properties of the hydrogel, while the loss modulus (G") describes its viscous properties. When the storage modulus of the hydrogel during deformation exceeds the loss modulus (i.e., G'>G"), it indicates that the hydrogel has sufficient cross-linked network structure to ensure its good elastic performance. On the contrary, if the loss modulus exceeds the storage modulus (i.e., G">G'), it indicates that under given stress conditions, the cross-linked network of the hydrogel is insufficient to maintain its gel state. Therefore, by comparing G' and G", the behavior and performance of the sample under different external stress stimuli can be effectively analyzed.

[0092] Use a 1mL syringe to transfer 300μL of HA-PB and drop it on the mold of the rotational rheometer (P20TiL, diameter 20mm). Quickly add 100μL of TA and PVA solutions in turn. Start the machine, and during the test, the plate spacing between the molds is 1mm, the temperature is 37℃, the frequency is 1Hz, and the test mode is time-modulus.

[0093] The precursors of HTP hydrogel were added to the mold in sequence. Under the set parameters, G' and G" of HTP hydrogel gradually increased with the passage of time. At 54 seconds, G'>G" indicated the formation of hydrogel.

[0094] Example 10

[0095] To evaluate the effect of HTP hydrogel on the viability of L. reuteri, L. reuteri was stained with green fluorescent nucleic acid stain SYTO 9, and L. reuteri@HTP hydrogel with a thickness of about 5 mm was placed in a confocal laser scanning microscope to observe the bacterial activity of L. reuteri. To verify the viability of L. reuteri in gastric juice, L. reuteri labeled with SYTO 9 and red fluorescent nucleic acid stain propidium iodide PI were placed in simulated gastric juice, incubated at 37°C for 24 hours, and the survival of L. reuteri was observed using an inverted fluorescence microscope.

[0096] like Figure 5 As shown in ab, it can be clearly observed that there are a large number of green signals (representing living bacteria) in the hydrogel, while the red signal (representing dead bacteria) is almost non-existent, and at a higher magnification, L. reuteri can be observed to be in the shape of slender rods, indicating that the vitality of L. reuteri is not affected.

[0097] The special physiological environment of the stomach makes it difficult for most microorganisms to survive there. L. reuteri was further stained with SYTO 9 / PI live / dead bacteria double staining reagent and incubated in SGF for 2 hours. Figure 5 As shown in c, a large number of L. reuteri still survived after the incubation, indicating that gastric juice has little effect on the vitality of L. reuteri, which makes it have the potential to treat HP.

[0098] Embodiment 11

[0099] In vitro antibacterial ability of L. reuteri@HTP hydrogel

[0100] Mixed antibiotics were prepared in advance. Specifically, 40 mg of polymyxin, 60 mg of trimethoprim, 40 mg of amphotericin B, and 50 mg of vancomycin were weighed into a sterile stoppered glass bottle, 150 mL of distilled water was added, and the volume was adjusted to 200 mL after stirring evenly. The mixture was stored in a refrigerator at 4°C for later use.

[0101] HP was scraped from the blood agar plate and resuspended in Brucella broth. The bacterial concentration was adjusted to 1 × 10 6 CFU / mL. Then, 10 6 CFU / mL of bacteria were incubated with 10 mL of Brucella broth, 1 mL of FBS, 50 μL of mixed antibiotics, and 1 mg of HTP hydrogel in a tri-gas incubator for 24 h under shaking. The plates were incubated for another 72 h, and then the colonies on the plates were photographed and counted.

[0102] HP solution was mixed with HTP (10 mg / mL), L. reuteri (1×10 8 After co-culture with L. reuteri@HTP (10 mg / mL), the mixed solution was added to SYTO 9 / PI live / dead bacteria double staining reagent and incubated under weak light for 15 minutes. The solution was then observed and photographed under an upright fluorescence microscope (MshOt, MF43-N, China), and the HP solution alone was used as the control group (Control).

[0103] like Figure 6 As shown in a and b, the HTP hydrogel without L. reuteri loading had poor anti-HP effect, while the inhibitory ability of the L. reuteri group and L. reuteri@HTP on HP was significantly enhanced (p<0.01). This indicates that the anti-HP effect of L. reuteri@HTP is almost entirely from L. reuteri. Live-dead staining of HP was performed, and similar results were observed as those of plate culture ( Figure 6 c).

[0104] Example 12

[0105] In vitro antioxidant capacity of HTP hydrogel

[0106] HP infection is a core link in the pathogenesis of gastritis. HP can cause inflammatory responses in gastric epithelial cells by releasing toxins such as CagA and VagA. In addition, HP infection also activates the host's immune system, causing a large number of inflammatory cells, especially macrophages and neutrophils, to gather at the site of infection, leading to abnormal oxidative stress responses. As mentioned earlier, TA is a highly efficient ROS scavenger that can effectively alleviate the oxidative stress response of tissues. The scavenging ability of HTP hydrogels for nitrogen-centered free radicals and oxygen-centered free radicals was tested using DPPH· and PTIO· free radical scavenging assays. TA has a strong electron donating ability. When encountering DPPH· and PTIO· free radicals, it can donate electrons to these free radicals, thereby stabilizing the free radicals and converting them into less active forms, which is specifically manifested in a decrease in the UV absorption intensity of the solution and a fading of the solution color. Figure 7 As shown in a, at a low concentration of 5 mg / mL, the color of the DPPH· free radical solution faded rapidly, and the scavenging rate reached 31.7±2.5% at 30 s, and the scavenging rate was close to the peak value of 90.9±1.2% at 90 s. However, the scavenging rate changed little over time, and the scavenging rate of HTP hydrogel for DPPH· free radicals was 94.7±0.4% at 180 s. The scavenging ability of HTP hydrogel for PTIO· free radicals was tested at a concentration of 20 mg / mL, as shown in Figure 7As shown in b, the PTIO free radical scavenging rate gradually increased over time, reaching 59.9±2.4% at 120 min. Next, in order to explore whether L. reuteri@HTP can scavenge cellular free radicals, RAW264.7 cells were treated with H2O2 to establish a cellular inflammation model. Subsequently, the cells were treated with PBS, Lactobacillus reuteri or L. reuteri@HTP, ROS were labeled with DCFH-DA as a probe, and the intracellular ROS level was detected by flow cytometry and cell fluorescence. Figure 7 As shown in Figure ch, compared with cells treated with H2O2 alone, the number of ROS-positive cells did not change significantly after the addition of L.reuteri, while the number of ROS-positive cells decreased from 60.6% to 7.24% after the addition of L.reuteri@HTP, which was comparable to untreated cells (healthy cells), indicating that L.reuteri@HTP has excellent ROS scavenging ability. The above test results show that HTP hydrogel has excellent ROS scavenging potential. The antioxidant capacity of HTP hydrogel was further verified by cell experiments. Figure 7 As shown in e, a clear shift to a higher mean fluorescence intensity was observed after 3 h of incubation.

[0107] Embodiment 13

[0108] Biosafety of hydrogels

[0109] First, the cell compatibility of the hydrogel was verified. Gastric epithelial cells were cultured at 1×10 4 The cells were seeded into 96-well plates at a density of 100 μL. After the cells attached overnight, the culture medium was replaced with 0, 5, 10, 15 and 20 mg / mL of L. reuteri@HTP hydrogel extract and cultured for 24 hours, 48 ​​hours and 72 hours, respectively. Cell viability was assessed using the cell counting kit-8 (CCK-8) assay. The culture medium was replaced with 100 μL of 10% CCK-8 dye solution per well. After incubation at 37°C for 1 hour, the cells were counted using a microplate reader (Molecular Devices i3, USA) was used to measure water-soluble methotrexate dye at a wavelength of 450 nm. Three replicate wells were used in each group, and the cell viability was calculated according to the following formula:

[0110]

[0111] Live / dead cell staining assay was performed by replacing the culture medium with calcein-AM and PI dyes after the materials were co-cultured with cells. After incubation at 37°C for 30 min, the cells were observed under a fluorescence inverted microscope.

[0112] The blood compatibility of the hydrogel was then verified. Fresh blood from rats was collected, centrifuged at 3000 rpm for 5 minutes, and the supernatant was removed. The collected red blood cells were washed three times with PBS, and then 1 mL of red blood cells was resuspended in 49 mL of PBS. Then, 500 μL of the red blood cell suspension was mixed with 500 μL of PBS containing different concentrations of L. reuteri@HTP (0, 5, 10, 15 and 20 μg / mL). As negative and positive controls, 500 μL of red blood cells were added to 500 μL of PBS and deionized water, respectively. The mixture was incubated at 37°C for 2 hours, and the absorbance of the supernatant was measured at a wavelength of 540 nm using a microplate reader. The hemolysis rate is calculated as follows:

[0113]

[0114] 200 mg of the hydrogel was implanted subcutaneously in mice, and the heart, liver, spleen, lung, and kidney of the mice were collected for H&E staining to assess animal safety. Blood was collected from the eyes of the mice for routine blood tests, and serum was collected for liver and kidney function tests.

[0115] like Figure 8 As shown in ad, at different concentrations, the gastric epithelial mucosal cells had good vitality and were stained green. Even on the third day, the extract of L. reuteri@HTP hydrogel still did not show obvious cytotoxicity. The CCK-8 assay was used for evaluation. The results showed that the survival rate of gastric epithelial mucosal cells in the extract of high concentration (20 mg / mL) of L. reuteri@HTP hydrogel was higher than 80%, indicating that L. reuteri@HTP hydrogel had no obvious inhibitory effect on cells.

[0116] In clinical trials, hydrogels inevitably have the risk of contact with blood, so the hemolysis rate of hydrogels must be controlled within a safe range, otherwise there will be potential safety hazards. When 2% v / v rat red blood cell suspension was co-incubated with L. reuteri@HTP hydrogels of different concentrations, it can be observed that the red blood cells in the control group with deionized water were ruptured, and the red blood cells could not be separated even after centrifugation. However, no obvious hemolysis occurred in the experimental group after the red blood cells were co-incubated with 20 mg / mL L. reuteri@HTP hydrogels ( Figure 8 e). The supernatant after incubation was tested under a UV spectrophotometer, and the hemolysis rates of L. reuteri@HTP hydrogels at different concentrations were calculated to be less than 5% ( Figure 8 f). According to the international standard of ISO 10993-4, L. reuteri@HTP hydrogel has good blood compatibility.

[0117] like Fig. 9As shown in a, all mice showed no obvious poisoning or inflammatory reaction. Blood samples were collected from the mice after the experiment for routine blood tests and blood biochemical tests, as shown in Fig. 9 As shown in Figure 2, no abnormal blood picture was found in the mice in all experimental groups. Therefore, based on the above experiments, it is reasonable to believe that L. reuteri@HTP hydrogel can be used in subsequent animal experiments as a safe biomaterial.

[0118] Embodiment 14

[0119] Cell migration assay

[0120] The cells were evenly plated in a 6-well plate and starved. A 10 μL pipette tip was used to scratch the cell monolayer. The cells were then rinsed three times with PBS to remove floating cells. Subsequently, the cells were co-cultured with HTP (10 mg / mL) or L. reuteri@HTP (10 mg / mL) at 37°C. Images were collected at 0, 12, 24, 36, and 48 hours. The calculation formula (3.4) for cell migration rate is as follows:

[0121]

[0122] like Fig.10 As shown in a, L. reuteri@HTP hydrogel significantly accelerated the migration rate of cells. Using Image J to calculate the blank area, the migration rates of cells incubated with L. reuteri@HTP hydrogel were 58.8±6.2% and 68.8±5.6% at 24h and 36h, respectively, while the migration rate of cells in the control group after 36h was 39.0±5.2% ( Fig.10 b), this is because HTP hydrogel is rich in HA, which can promote adhesion molecules that affect the cell surface, such as integrins, and regulate the adhesion and detachment of cells and extracellular matrix. At the same time, HA can also interact with receptors on the cell surface (such as CD44), activate internal signal transduction pathways, and promote cell proliferation and differentiation. This shows that L. reuteri@HTP hydrogel has a good effect in promoting gastric mucosal repair.

[0123] Embodiment 15

[0124] ROS-responsive degradation experiment of hydrogel

[0125] L. reuteri@HTP was labeled with Cy7 (MCE, HY-D0825), and 500 μL of Cy7-labeled L. reuteri@HTP was gavaged into normal mice and HP-infected mice (n=5). Ten hours later, the stomach of the mice was imaged using an IVIS imaging system (TanonABL X6, China).

[0126] By labeling L. reuteri, the degradation and release of L. reuteri from L. reuteri@HTP hydrogel in mice were observed ( Fig.11 ab). Compared with healthy mice, the fluorescence intensity in the stomach of HP-infected mice was significantly increased (p<0.0001), and the fluorescence intensity was increased by about 38 times compared with healthy mice. This indicates that due to the influence of inflammation, the higher ROS concentration promoted the accelerated degradation of L.reuteri@HTP hydrogel in the stomach of HP-infected mice, and a large amount of L.reuteri was released and retained in the stomach, while only a small amount of L.reuteri remained in the stomach of healthy mice. This shows that L.reuteri@HTP hydrogel can responsively and efficiently release L.reuteri in HP-infected mice, improving the therapeutic effect.

[0127] Example 16

[0128] Establishment of HP infection model in vivo

[0129] Six-week-old C57BL / 6 mice were provided by Jiangsu Jidacheng Pharmaceutical Biotechnology Co., Ltd. All mice were orally administered 300 μL of HP suspension (1×108 CFU / mL) once every other day for two consecutive weeks. After infection, the mice were housed under normal conditions for four weeks. Subsequently, five mice were randomly selected for urease test to evaluate the infection status.

[0130] In vivo evaluation of therapeutic efficacy of HP infection and intestinal flora sequencing

[0131] The HP-infected mice were randomly divided into six groups (n=8), including PBS group, HTP group, L. reuteri group, antibiotic group (clarithromycin, 14.3 mg / kg / day) and L. reuteri@HTP group. In addition, a control group that was not infected with HP and treated with PBS was included for comparison with the above five groups.

[0132] Each group received 1×10 8 CFU / mL HP (PBS was used for the uninfected control group) was inoculated seven times. After four weeks of colonization and infection, five mice were randomly selected and killed, and the successful establishment of the HP infection mouse model was verified by rapid urease detection method ( Fig.12 a). Then, mice were given oral gavage treatment once a day for one week, and the mice were killed on the second day after the treatment. HP infection in the stomach of mice was observed by plate coating and H&E staining. Fig.12As shown in bc, the blood agar plate results showed that the antibiotic group and the LR@HTP hydrogel group had fewer HP colonies. The colony count showed that compared with the PBS control group, the number of HP in the stomach of the HTP group did not change significantly, and the HP in the LR group was significantly reduced. The number of HP colonies in the LR@HTP hydrogel group was not significantly different from the positive control group treated with antibiotics and was significantly lower than that in the PBS group (p<0.00001). In the H&E stained sections, we can observe curved rod-shaped HP colonization (red arrows), and after LR@HTP hydrogel treatment, the number of HP in the stomach was significantly reduced and there was no difference from the antibiotic treatment group ( Fig.12 c).

[0133] To evaluate the in vivo antibacterial capacity of the hydrogel, all mice were sacrificed the day after the end of treatment and plate-spread to assess the in vivo antibacterial effect.

[0134] The severity of gastritis was evaluated from the histological changes of HP mice (HP, chronic inflammatory lesions, activity, atrophy, and intestinal metaplasia), and the degree of each histological change was evaluated and divided into 4 levels: none, mild, moderate, and severe. The in vivo anti-inflammatory effect of the hydrogel was evaluated by enzyme-linked immunosorbent assay (ELISA) analysis and immunohistochemistry analysis. According to the instructions of the ELISA kit, the treated mouse serum was diluted and mixed with the ELISA detection solution (MPO activity, IL-10, IL-6, IL-1β, etc.). The absorbance was measured at OD = 450nm. After stopping the administration, the gastric tissue of the mouse was collected for immunohistochemistry analysis. The tissue was fixed, dehydrated with formaldehyde, and embedded in paraffin.

[0135] To evaluate the mucosal repair in mice. The stomachs of treated mice, HP-infected mice, and normal mice were collected to observe the repair of gastric tissue inflammation under optical conditions. Fluorescence staining was also performed to evaluate the repair of gastritis in vivo. Ki67 and β-catenin staining were used to evaluate the apoptosis of gastric epithelial cells, and ZO-1 and Occludin-1 staining were used to evaluate the repair of gastric mucosa. Fecal samples from each group of mice after treatment were collected for intestinal microbiota analysis. The abundance and diversity of bacteria in mouse feces were determined by 16S rRNA sequencing.

[0136] like Fig.13 By specific labeling of cell membrane and other subcellular structures, significant differences in the expression of intercellular adhesion protein (β-catenin), proliferating cell nuclear antigen (PCNA), tight junction protein Occludin and Claudin-1 were observed among the treatment groups. DAPI was used for nuclear staining to show the nuclear localization of cells.

[0137] In terms of β-catenin expression, the control group showed a uniform cell adhesion interface and cytoplasmic distribution. In contrast, the L. reuteri@HTP-treated group showed a significant increase in β-catenin expression, indicating the strengthening of intercellular junctions and the promotion of cell adhesion and proliferation. The antibiotic-treated group showed a significantly reduced expression of β-catenin, which may reflect the impairment of cell junctions and signal transduction under this condition. PCNA, a marker of cell proliferation, was significantly enhanced in the HTP and L. reuteri@HTP groups, indicating the activation of gastric mucosal cell cycle activity and the significant increase in proliferation potential under this condition, which may be related to the biological activity of HA. In addition, the staining results of Occludin and Claudin-1 showed that the integrity of the tight junction structure of the mucosa was enhanced in the L. reuteri and L. reuteri@HTP-treated groups, indicating that this treatment helps to restore and maintain the barrier function of the gastric mucosa.

[0138] Evaluating the therapeutic effect of gastritis with immunohistochemistry

[0139] To further understand the alleviating effect of L. reuteri@HTP hydrogel on gastritis, gastritis in HP mice was evaluated. Fig.14 As shown in a, the results of H&E staining sections of gastric tissue after treatment in different experimental groups showed that a large number of inflammatory cells were observed in the gastric tissue of mice infected with Helicobacter pylori (PBS group). After L. reuteri and antibiotic treatment, the infiltration of inflammatory cells in gastric tissue was reduced and located on the surface. After L. reuteri@HTP treatment, the infiltration of inflammatory cells in gastric tissue was significantly reduced. In addition, the inflammation in H&E staining of gastric tissue was scored according to the Sydney system score. Compared with the PBS group, the gastric inflammation score of HP mice treated with L. reuteri@HTP hydrogel was significantly reduced (p<0.05). The above results show that L. reuteri@HTP hydrogel can effectively relieve gastric inflammation in mice while eradicating HP in the stomach.

[0140] Subsequent cytokine analysis ( Fig.14 bj) shows the expression levels of various immunomodulatory factors under different treatment conditions. Specifically, MPO activity ( Fig.14 b) IL-18 Fig.14 e) and IL-1β ( Fig.14 d) As a key pro-inflammatory cytokine, its level was significantly decreased in the L. reuteri@HTP-treated group, indicating that the gastric inflammatory response was alleviated. Fig.14 f), which is usually associated with Th17 cell-mediated inflammatory response, was reduced after L. reuteri@HTP treatment, which may involve the inhibition of specific inflammatory pathways. Fig.14 i) IL-12 ( Fig.14 h) and IL-2 ( Fig.14 g) suggest potential regulation of Th2 and Th17 cell responses. In addition, TGF-α ( Fig.14 c) indicates the promotion of tissue repair and possible fibrosis, while IL-10 ( Fig.14 j) The increase in levels reflects the strong anti-inflammatory response and immune regulation induced by L. reuteri@HTP treatment.

[0141] Effect of L. reuteri@HTP hydrogel on intestinal microbiota homeostasis

[0142] To verify the effect of L. reuteri@HTP on intestinal flora, the intestinal flora of different treatment groups was analyzed using 16S rRNA technology, and the diversity index, community distance matrix and relative abundance were used to characterize the microbial diversity and composition. Fig.15 As shown in Figure ac, compared with the antibiotic group used in traditional therapy, the Chao1 index results showed that L. reuteri@HTP significantly increased the species richness of microorganisms (p<0.0001), while the Shannon index characterized the diversity and uniformity of the community, and a significant increase was also observed in the L. reuteri@HTP-treated group (p<0.0001). The Simpson index reflects the uniformity and dominance of species. There was no significant difference between the mice treated with L. reuteri@HTP hydrogel and the control group, suggesting that the dominance of species was not changed due to L. reuteri@HTP treatment. The weighted UniFrac distance was further used to measure the difference in community structure ( Fig.15 d), it was found that L. reuteri@HTP treatment induced a significant shift in community composition. In addition, principal coordinate analysis (PCoA, Fig.15 e) and non-metric multidimensional scaling (NMDS, Fig.15 f) The results all confirmed the significant changes in the microbial community structure after L. reuteri@HTP treatment, reflecting that the spatial distribution of intestinal microbial diversity was significantly affected. Relative abundance analysis ( Fig.15 gh) further confirmed that the relative abundance of the genus Lactobacillus was significantly increased in the L. reuteri@HTP treatment group, which may be related to the direct introduction of L. reuteri and the bioscaffolding effect provided by HA. As a multifunctional polysaccharide, HA may promote the colonization and proliferation of L. reuteri in the intestine by maintaining water balance and providing extracellular matrix. In addition, the biocompatibility and immunomodulatory properties of HA may provide a more favorable intestinal microenvironment for L. reuteri, thereby optimizing the composition and function of the microbial community.

[0143] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the creative concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A ROS-sensitive hydrogel, characterized in that Includes aminophenylboronic acid, N-hydroxysuccinimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, hyaluronic acid, tannic acid and water.

2. The hydrogel according to claim 1, characterized in that Also included are polyvinyl alcohols.

3. The hydrogel according to claim 1, characterized in that Also included is Lactobacillus reuteri.

4. A method for preparing a ROS-sensitive hydrogel, characterized in that: include: A. Dissolve 0.5-2 g of HA in 100 mL of deionized water, add 0.5-1.5 g of aminophenylboronic acid, 0.1-1 g of N-hydroxysuccinimide and 0.1-1 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and stir evenly at room temperature to obtain a yellow viscous solution; dialyze the hyaluronic acid (HA) in deionized water to remove unreacted aminophenylboronic acid, N-hydroxysuccinimide and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; after the dialysis, freeze-dry the product to obtain HA-PB; B. Evenly mix the HA-PB aqueous solution and the tannic acid aqueous solution and stir to obtain a hydrogel.

5. The preparation method according to claim 4, characterized in that: In step B, the w / v concentration of the HA-PB aqueous solution is 4%, the w / v concentration of the tannic acid is 4%, and the volume ratio of the reaction of the HA-PB solution and the tannic acid solution is 3:

1.

6. The preparation method according to claim 4 or 5, characterized in that: It is characterized in that Step B also includes adding polyvinyl alcohol to the hydrogel.

7. The preparation method according to claim 6, characterized in that: The method also includes first dissolving polyvinyl alcohol in water, wherein the w / v concentration of the polyvinyl alcohol is 2%, and the volume ratio of the HA-PB solution to the polyvinyl alcohol solution is 3:(0.5-2).

8. The preparation method according to claim 4, characterized in that: Step B also includes adding L. reuteri to the HA-PB solution, wherein the amount of L. reuteri is (0.5-2)×10 8 CFU / mg.

9. The hydrogel according to any one of claims 1 to 3 is used to remove ROS or / and Inhibit the activity of HP and / or Repair gastric mucosa and / or Application in stabilizing intestinal flora.

10. The hydrogel prepared by the preparation method according to any one of claims 4 to 8 is effective in removing ROS or / and, Inhibit the activity of HP and / or Repair gastric mucosa and / or Application in stabilizing intestinal flora.

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