Suction-excited hydrogel with regeneration activity and preparation method of mucus-excited hydrogel

Abstract

The invention belongs to the technical field of biomedicine, and discloses mucus excitation hydrogel with regeneration activity and a preparation method thereof, the mucus excitation hydrogel is composed of dihydrocaffeic acid modified boloxamide, hyaluronic acid, epigallocatechin-3-gallate and bioactive factors, and the bioactive factors are KPV tripeptide and epidermal growth factors. The PHE-EK is a flowable liquid at room temperature and is gelled within 10 seconds at the body temperature. And the strain of the PHE-EK hydrogel is 77.8%. The PHE-EK hydrogel specifically adheres to the inflammatory colon through electrostatic interaction. The PHE-EK solution provided by the invention has good temperature sensitivity. The PHE hydrogel has relatively high mechanical strength and relatively high fracture strain. The PHE hydrogel can specifically adhere to the inflammatory colon by electrostatic interaction. PHE-EK enema is a promising method for treating ulcerative colitis.

Description

A mucus-induced hydrogel with regenerative activity and its preparation method Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a mucus-induced hydrogel with regenerative activity and its preparation method. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory disease affecting the colorectal region, with an increasing incidence worldwide. UC patients present with recurrent inflammation and ulcers limited to the superficial mucosa of the colorectal region. Mainstream treatments for UC include 5-aminosalicylic acid, steroids, or immunosuppressants to alleviate inflammatory symptoms. Additionally, antibiotics (metronidazole or ciprofloxacin) are sometimes used as adjunctive therapy to control bacterial infections. Currently, new understanding of the pathogenesis of UC has led to the development of antibody-based treatments. For example, anti-TNFα antibodies can effectively combat mild to severe colitis; however, almost all available antibody-based therapies work by suppressing the systemic immune response or blocking specific inflammatory molecules. Due to systemic immunosuppression, these treatment regimens carry the risk of developing infectious or neoplastic diseases.

[0003] The intestinal mucosal barrier, composed of mucus, epithelial cells, and tight junction proteins (TJs), plays a crucial role in intestinal homeostasis. Mucus, a viscoelastic hydrogel, is a first-line barrier protecting the gastrointestinal tract (GI) from harmful microorganisms and antigens. Colonic mucus, with its gel-like bilayer structure and antibacterial activity, serves as the first line of innate host defense. Colonization of the symbiotic gut microbiota is confined to the outer, “loose” mucus layer; while the “inner” adhering mucus is highly dense and devoid of bacteria due to its richness in antimicrobial peptides. A disrupted intestinal mucosal barrier allows pathogens, allergens, or luminal toxins to penetrate the lamina propria, home to innate immune cells, triggering an immune-related inflammatory response. Mucus loss and crypt epithelial cell collapse have been shown to be associated with the pathogenesis of colitis. Intestinal mucosal healing is currently considered the endpoint of UC treatment because it enables sustained remission and resection-free survival. Barrier therapy (BT), a physical, non-pharmacological modality for managing clinical syndromes without compromising systemic immune responses, is attractive to UC patients. For example, coliforms... One barrier therapy approved in Europe is a polysulforaphane enema solution that forms a viscous, paste-like barrier on the diseased colon of UC patients. Additionally, a molecular coating composed of o-nitrobenzaldehyde (NB)-modified gelatin has been shown as a barrier therapy to form a thin biophysical barrier on the intestinal surface through covalent interactions, isolate intestinal irritant metabolites, and modulate intestinal microbial homeostasis in the treatment of UC in mice. However, these barrier therapies, as transient interventions, generally do not heal the damaged intestinal mucosa well.

[0004] Due to their mucus-like networks, polymeric hydrogels and growth factors have also been developed as barrier therapies for healing colonic mucosa in the treatment of colitis. In previous studies, a thermosensitive hydrogel of caffeic acid dihydrate-modified doloxamine (DAP) was developed for rectal delivery of epidermal growth factor (EGF). However, the DAP hydrogel struggled to specifically adhere to the inflamed colon, unlike healthy patches. Along with the loss of colonic mucus, positively charged components, e.g., bactericidal proteins, or antimicrobial peptides, aggregate on the inflamed colonic mucosa. Therefore, the specific retention of negatively charged hydrogels on the inflamed intestine has been widely used in the treatment of UC. For example, negatively charged hyaluronic acid microsphere hydrogels aggregate on the inflamed colon, modulating the gut immune microbiota microenvironment. Furthermore, negatively charged dual-network γ-polyglutamic acid hydrogels can specifically deliver anti-inflammatory tripeptides (KPV) for the treatment of TNBS-induced colitis. However, due to their limited diffusion capacity, these prepared hydrogels struggle to penetrate proximal colitis. Moreover, these treatments are not effective against pathogenic bacterial infections.

[0005] Based on the above analysis, the problems and shortcomings of the existing technology are as follows:

[0006] (1) Existing antibody-based treatments carry the risk of inducing infectious or neoplastic diseases. Existing barrier therapies are mainly used as short-term interventions and are usually not effective in healing damaged intestinal mucosa.

[0007] (2) Some hydrogel formulations (such as dihydrocaffeic acid-modified doloxaamine thermosensitive hydrogel) can deliver growth factors, but they are difficult to specifically adhere to the inflamed colonic mucosa. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides a mucus-inducing hydrogel with regenerative activity and its preparation method.

[0009] The present invention is achieved as follows: a mucus-inducing hydrogel with regenerative activity, wherein the mucus-inducing hydrogel with regenerative activity is composed of dihydrocaffeic acid-modified poloxamine (a colitis-specific adhesive), hyaluronic acid, epigallocatechin-3-gallate (an antibacterial agent), and bioactive factors, wherein the bioactive factors are KPV tripeptide and epidermal growth factor.

[0010] Furthermore, PHE-EK is a flowable liquid at room temperature and gels within 10 seconds at body temperature.

[0011] Furthermore, the strain of the PHE-EK hydrogel is 77.8%.

[0012] Furthermore, the PHE-EK hydrogel specifically adheres to the inflamed colon through electrostatic interactions.

[0013] Furthermore, after PHE-EK treatment, epithelial cells were well-aligned, and tight junction proteins (ZO1 and Claudin-5) were significantly upregulated; PHE-EK also promoted non-fibrotic remodeling of the colonic extracellular matrix by upregulating the collagen-iii / collagen-i ratio and reducing the expression of MCP1 and α-SMA.

[0014] Another objective of this invention is to provide a method for preparing a mucus-inducing hydrogel with regenerative activity. The method for preparing the mucus-inducing hydrogel with regenerative activity includes: integrating temperature-sensitive dihydrocaffeic acid-modified doloxamine (DAP) with antibacterial components EGCG and hyaluronic acid (HA) to prepare an in-situ hydrogel PHE; and adding KPV and EGF to growth factors to form the PHE hydrogel, thereby generating a mucus-inducing hydrogel PHE-EK.

[0015] Furthermore, a PHE solution is prepared using a gradient dissolution method, specifically including the following steps:

[0016] Step 1: Dissolve HA powder (1.0g) in distilled water (100mL);

[0017] Step 2: Dissolve DAP powder (20.0g) in a cold HA solution at 4°C and stir gently;

[0018] Step 3: Further dissolve EGCG (1.0g) to obtain a malignant PHE solution;

[0019] Step 4: Dissolve EGF, KPV, or both directly in a cold PHE solution to prepare a PHE-EK solution.

[0020] Another object of the present invention is to provide an application of a mucus-stimulating hydrogel with regenerative activity in the treatment of ulcerative colitis.

[0021] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows:

[0022] First, regarding the technical problems existing in the aforementioned prior art and the difficulty of solving these problems, the creative technical effects that arise after solving the problems are described in detail below:

[0023] PHE-EK exhibits good thermosensitivity, remaining a flowable liquid at room temperature and gelling within 10 seconds at body temperature. The PHE-EK hydrogel possesses good mechanical strength with a strain of 77.8%. It also demonstrates antibacterial activity against *Escherichia coli*. Importantly, in vitro and in vivo adhesion assays show that the PHE-EK hydrogel specifically adheres to the inflamed colon via electrostatic interactions. When administered rectally as a biomimetic mucus to rats with colitis, PHE-EK effectively inhibited weight loss, reduced the disease activity index, and improved colonic shortening. This PHE-EK treatment significantly alleviated TNBS-induced colitis in mice.

[0024] Furthermore, PHE-EK treatment significantly inhibited colonic ulcerative colitis in rats by increasing the levels of pro-inflammatory cytokines (e.g., IL-1β, IL-6, and TNF-α) in the lamina propria or epithelial cells. In addition, PHE-EK treatment resulted in well-aligned epithelial cells and significant upregulation of tight junction proteins (ZO1 and Claudin-5). PHE-EK also promoted non-fibrotic remodeling of the colonic extracellular matrix by upregulating the collagen-iii / collagen-i ratio and decreasing the expression of MCP1 and α-SMA. In conclusion, PHE-EK enema is a promising approach for the treatment of ulcerative colitis.

[0025] Secondly, this invention has made significant technological progress in material design, biocompatibility, targeted therapy and tissue repair, solved key technical problems encountered by traditional hydrogels in biomedical applications, and demonstrated great application potential.

[0026] Enhanced biocompatibility and activity: The unique composition of this hydrogel enhances its compatibility and activity with organisms, which is beneficial for promoting the repair and regeneration of damaged tissues.

[0027] Precise targeting and effective treatment: By specifically adhering to the inflamed area through electrostatic interaction, more precise targeted treatment is achieved, improving treatment efficiency and effectiveness.

[0028] Optimized stability and manufacturability: The gelation properties at different temperatures make the hydrogel easy to maneuver and stable in in vivo and in vitro environments.

[0029] Promotes extracellular matrix remodeling: By regulating the expression of related proteins and inhibiting inflammatory factors, it effectively promotes the non-fibrotic remodeling of damaged tissues, which helps improve tissue repair and functional recovery. Attached Figure Description

[0030] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments of the present invention will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1 is a schematic diagram of the mucus-induced PHE-EK hydrogel as a physical barrier for UC treatment provided in an embodiment of the present invention.

[0032] Figure 2 is a flowchart of the method for preparing PHE solution using gradient dissolution method provided in an embodiment of the present invention;

[0033] Figure 3 is a schematic diagram of the properties of the PHE hydrogel provided in the embodiments of the present invention: (A) solution-gel transition of PHE hydrogel at 4℃ or 37℃; (B) PHE hydrogel; (C) viscosity curve of PHE hydrogel; (D) oscillation temperature scan of PHE hydrogel; (E) oscillation frequency scan of PHE hydrogel; (F) oscillation strain scan of PHE hydrogel; (G) viscosity of PHE hydrogel at shear rate; (H) self-healing of PHE hydrogel at low strain (1%) or high strain (100%); (I) ability of PHE hydrogel to resist dilution beyond warm water (37℃); (J) SEM image of PHE hydrogel (data are expressed as mean ± SEM; ***P<0.001, **P<0.01, *P<0.05; n=3);

[0034] Figure 4 is a schematic diagram of the in vitro adhesion strength of PHE hydrogel to tissue or artificial PVA film matrix provided in the embodiments of the present invention: (A) Schematic diagram of the overlap-shear adhesion test of a general testing machine; (B) Stress-strain curve of PHE hydrogel on rat skin; Quantitative analysis of adhesion force (C) and adhesion energy (D) based on stress-strain curve; Stress-strain curve of PHE hydrogel on neutral (E), positive (F), and negative (G) substrates; Quantitative analysis of adhesion energy (H) and adhesion force (I) (data are mean ± standard error; ***P<0.001, **P<0.01, *P<0.05; n=3);

[0035] Figure 5 is a schematic diagram of the in vitro antibacterial activity of the PHE hydrogel provided in the embodiments of the present invention: (A) diffusion plate method to show the antibacterial activity of PHE hydrogel; (B) colony qualitative analysis; (C) vibration method to detect the antibacterial effect of PHE hydrogel; (D) schematic diagram of bacterial migration; (E) optical density (OD) at 600 nm in the upper / lower cavity (data are expressed as mean ± standard error; ***P<0.001, **P<0.01, *P<0.05, n=3);

[0036] Figure 6 is a schematic diagram of the in vivo adhesion of 5PHE hydrogels on inflamed colon provided in this embodiment of the invention: (A) Experimental protocol for adhesion of PHE hydrogels on colitis colonic mucosa after PBS washing; (B) In vitro imaging of the remaining PHE hydrogels on the colon after PBS washing; (C) In vivo abdominal imaging of TNBS-induced colitis rats or healthy rats after rectal administration; (D) Fluorescence distribution of PHE hydrogels on colonic tissue 8 hours after rectal administration; (E) Quantitative analysis of the remaining PHE hydrogels based on in vitro imaging; (F) Quantitative analysis of the remaining PHE hydrogels based on in vivo imaging; (G) Quantitative analysis of the remaining PHE hydrogels based on colonic tissue imaging (data are expressed as mean ± standard error; *P<0.05, n=3).

[0037] Figure 7 is a schematic diagram of the effective relief of TNBS-induced colitis in rats by PHE-EK provided in the embodiments of the present invention: (A) TNBS-induced colitis regimen and PHE-EK treatment regimen; (B) weight loss of rats with colitis treated with different enemas and (C) DAI score; (D) representative colon images; (E) statistical length of colon after different treatments; (F) spleen weight in each group; (G) endoscopy images of colonic mucosa after different treatments; (H) H&E, AB-PAS, and Masson staining (data are expressed as mean ± standard error; ***P<0.001, **P<0.01, *P<0.05, compared with the TNBS group; n=5);

[0038] Figure 8 is a schematic diagram of how PHE-EK reduces the expression of pro-inflammatory cytokines provided in the embodiments of the present invention: (A) IHC staining of pro-inflammatory cytokines (IL-6, TNFα, IL1β) and anti-inflammatory cytokines (IL10); (B) Quantitative analysis of inflammatory cytokines based on IHC staining (data are mean ± standard error; ***P<0.001, **P<0.01, *P<0.05, n=5);

[0039] Figure 9 is a schematic diagram of the repair of the colonic mucosal barrier after PHE-EK treatment provided in the embodiments of the present invention: (A) is the immunofluorescence staining of tight junction proteins (ZO1 and Claudin-5), and (B) is the quantitative analysis of TJs based on immunofluorescence staining (data are mean ± standard error of mean; ***P<0.001, **P<0.01, *P<0.05, n=5);

[0040] Figure 10 is a schematic diagram of the inhibition of colonic fibrosis by colon after PHE-EGF / KPV enema treatment provided in the embodiment of the present invention: MCP1, α-SMA, i collagen and iii collagen (A) IHC staining; (B) IHC staining quantitative analysis (data are mean ± standard error; ***P<0.001, **P<0.01, *P<0.05, n=5). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0042] Two specific application embodiments of the present invention are as follows:

[0043] Example 1: Treatment of Inflammatory Bowel Disease

[0044] Used in the treatment of inflammatory bowel diseases such as Crohn's disease or ulcerative colitis.

[0045] Targeted adhesion: The hydrogel rapidly gels at body temperature and adheres specifically to the inflamed colon through electrostatic interactions.

[0046] Bioactive release: Contains bioactive factors (KPV tripeptide and epidermal growth factor), which are slowly released after the hydrogel solidifies, promoting the repair and regeneration of damaged tissues.

[0047] Tissue remodeling: By regulating collagen ratios and inhibiting inflammatory factors (such as MCP1 and α-SMA), hydrogels promote non-fibrotic remodeling of the extracellular matrix in colon cells, improving tissue functional recovery.

[0048] Example 2: Promoting skin wound healing

[0049] Used to treat burns, chronic ulcers, or other skin wounds.

[0050] Forming a protective layer: At body temperature, the hydrogel forms a stable gel layer that covers the skin wound, forming a physical barrier that protects the wound from external microorganisms.

[0051] Continuous release of bioactive factors: Bioactive factors (such as epidermal growth factor) in the hydrogel are slowly released at the wound site, promoting the growth and differentiation of new cells and accelerating the wound healing process.

[0052] Promotes cell remodeling and healing: Hydrogels help restore cell structure and function in the wound area by providing a suitable moist environment and regulating the composition of the extracellular matrix, thus promoting rapid wound healing.

[0053] This invention addresses the following problems and deficiencies in the prior art, achieving significant technological advancements:

[0054] Limited biocompatibility and bioactivity: Traditional hydrogel materials may lack sufficient biocompatibility and bioactivity, which limits their application in the biomedical field.

[0055] Insufficient precise targeting of inflamed sites: Many existing hydrogels have limited effectiveness in targeting and adhering to inflamed sites, which reduces treatment efficacy.

[0056] Operability and stability issues: Some hydrogels have insufficient stability and operability in in vivo and in vitro environments, which may lead to difficulties in use and poor results.

[0057] Effectiveness of extracellular matrix remodeling: Existing materials may have limited effectiveness in promoting non-fibrotic remodeling of damaged tissues, particularly the extracellular matrix.

[0058] To address the problems existing in the prior art, the technical solution adopted in this invention is as follows:

[0059] Material composition: Poloxamine modified with dihydrocaffeic acid, hyaluronic acid, epigallocatechin-3-gallate, and bioactive factors (KPV tripeptide and epidermal growth factor).

[0060] Temperature sensitivity and gelation: PHE-EK is a flowing liquid at room temperature, but it gels rapidly at body temperature, improving ease of use and stability.

[0061] Adhesion and targeting: PHE-EK hydrogel adheres specifically to the inflamed colon through electrostatic interactions, improving the effectiveness of targeted therapy.

[0062] Extracellular matrix remodeling promotion: PHE-EK promotes non-fibrotic remodeling of the colonic extracellular matrix by regulating collagen ratios and inhibiting inflammatory factors.

[0063] As shown in Figure 1, mucus-induced PHE-EK hydrogel serves as a physical barrier for UC treatment.

[0064] This invention provides a mucus-inducing hydrogel with regenerative activity, which is composed of dihydrocaffeic acid-modified poloxamine (a colitis-specific adhesive), hyaluronic acid, epigallocatechin-3-gallate (an antibacterial agent), and bioactive factors, namely KPV tripeptide and epidermal growth factor.

[0065] Furthermore, PHE-EK is a flowable liquid at room temperature and gels within 10 seconds at body temperature.

[0066] Furthermore, the strain of the PHE-EK hydrogel is 77.8%.

[0067] Furthermore, the PHE-EK hydrogel specifically adheres to the inflamed colon through electrostatic interactions.

[0068] Furthermore, after PHE-EK treatment, epithelial cells were well-aligned, and tight junction proteins (ZO1 and Claudin-5) were significantly upregulated; PHE-EK also promoted non-fibrotic remodeling of the colonic extracellular matrix by upregulating the collagen-iii / collagen-i ratio and reducing the expression of MCP1 and α-SMA.

[0069] This invention provides a method for preparing a mucus-inducing hydrogel with regenerative activity. The method includes: integrating temperature-sensitive dihydrocaffeic acid-modified doloxamine (DAP) with antibacterial components EGCG and hyaluronic acid (HA) to prepare an in-situ hydrogel PHE; and adding KPV and EGF to growth factors to form the PHE hydrogel, thereby generating a mucus-inducing hydrogel PHE-EK.

[0070] As shown in Figure 2, the PHE solution is prepared using a gradient dissolution method, which includes the following steps:

[0071] Step 1: Dissolve HA powder (1.0g) in distilled water (100mL);

[0072] Step 2: Dissolve DAP powder (20.0g) in a cold HA solution at 4°C and stir gently;

[0073] Step 3: Further dissolve EGCG (1.0g) to obtain a malignant PHE solution;

[0074] Step 4: Dissolve EGF, KPV, or both directly in a cold PHE solution to prepare a PHE-EK solution.

[0075] 1. Experimental Indicators

[0076] Synthetic dihydrocaffeic acid-modified doloxamine (DAP) was used. Hyaluronic acid (HA, 120 kDa), EGCG, and 2,4,6-trinitrobenzenesulfonic acid solution (TNBS, 5% w / v / 50% ethanol) were purchased from Aladdin Biochemical Technology (Shanghai, China). Recombinant human EGF was purchased from PeproTech (GMP 10015, Rocky Hills, USA). KPV was obtained from GL Biochemicals (Shanghai, China). RPMI 1640 medium and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA, USA). MTT and hydrogen peroxide kits were provided by 3Solar Biotechnology Co., Ltd. (Beijing, China).

[0077] 2. Preparation of PHE hydrogel

[0078] PHE solutions were prepared using a gradient dissolution method. Simply put, HA powder (1.0 g) was first dissolved in distilled water (100 mL). Then, DAP powder (20.0 g) was dissolved in the cold HA solution at 4°C with gentle stirring. Finally, EGCG (1.0 g) was further dissolved to obtain the malignant PHE solution. The final concentrations of DAP, HA, and EGCG in the PHE solution were 20%, 1%, and 1%, respectively. As controls, 20% poloxamer 407 (POL) or DAP solutions were also prepared. The gel time of the PHE solution was determined using the vial tilting method. PHE-EGF, PHE-KPV, and PHE-EK solutions were prepared by directly dissolving EGF, KPV, or both in the cold PHE solution.

[0079] 3. Viscosity and Rheology of PHE Hydrogel

[0080] The viscosity of the PHE solution was measured using a No. NDJ-8S rheometer with one rotor and a small sample adapter, at a temperature range of 15–45 °C, an angular frequency of 10 rad / s, and a heating rate of 1 °C / min. Furthermore, the rheology of the PHE hydrogel was performed on a DHR-2 rheometer (TA, USA) using a cone plate with a diameter of 25 mm (25 μm). Oscillatory temperature scans were conducted at temperatures ranging from 15 °C to 45 °C, an angular frequency of 10 rad / s, and a heating rate of 1 °C / min. The gel temperature was used as the point of intersection of the storage modulus (G) and loss modulus (G) curves. At 37 °C, the strain was 1%. Oscillatory strain scans were performed at a frequency of 37 °C and an angular frequency of 10 rad / s.

[0081] 4. Morphology of PHE hydrogel

[0082] PHE hydrogels were frozen in liquid nitrogen and then freeze-dried. Cross-sections of the freeze-dried PHE hydrogels were prepared and sputtered with gold at an accelerating voltage of 20 kV for scanning electron microscopy analysis (Hitachi S-800, Japan). Freeze-dried POL or DAP hydrogels were used as controls for comparative studies.

[0083] 5. Adhesion test of PHE hydrogel to tissue

[0084] The adhesion of PHE hydrogel to mucosal tissue was evaluated using a universal testing machine (UTM, EZ-SXSTD, Shimadzu, Japan). Simply put, two fresh colon pieces were glued to a glass slide, and then PHE hydrogel (200 μL) was applied to the surface of one mucosa, overlapping it with the other. Testing was conducted on the universal testing machine at a cross-head rate of 5 mm / min and a 20 N load element until complete separation. All tests were performed under a constant temperature of 37 °C. Displacement curves were plotted, and adhesion forces and energies were calculated. Furthermore, the electrostatic adhesion of PHE hydrogel to charged substrates was investigated using several modification methods described in previous studies. Simply put, three types of polyvinyl alcohol (PVA) films (positive, neutral, or negatively charged) were prepared by adding chitosan and poly(γ-glutamic acid). The adhesion of PHE hydrogel to these PVA films was also tested. At least three parallel experiments were performed for each sample.

[0085] 6. Antibacterial activity of PHE hydrogel

[0086] Escherichia coli ATCC8739 (E. coli) was used as an opportunistic pathogen. The antibacterial effect of PHE hydrogel was determined using the plate spread method. Simply put, 100 μL of E. coli, 1.0 × 10⁴ g / mL... 5 CFU / mL was inoculated onto agar plates. Then, 100 μL of cold PHE solution was evenly spread onto the agar plates and incubated at 37°C. After 12 h of incubation, bacterial clones were imaged, and the clone number was calculated using ImageJ software. Additionally, the antibacterial activity of PHE was detected using a shaking method. Simply put, 100 μL of PHE solution was first dissolved in 4 mL of TSB culture solution. One method involved E. coli cultured overnight. The E. coli suspension was added to the TSB solution to obtain approximately 10 E. coli at 5 CFU / mL. After shaking incubation at 37°C for 24 h, the optical density at 660 nm (OD660) was measured using a SpectraMax reader. As a control, the antibacterial activity of POL or DAP solutions was also tested. Furthermore, bacterial migration within the PHE hydrogel was detected using the Transwell method. Simply put, 0.5 mL of PHE hydrogel was evenly spread onto a 5.0 μm pore size polycarbonate resin transporous membrane. E. Dilute 1 OD / mL of E. coli with LB medium (1:1000), then dilute E. Add 200 μL of E. coli to the top. Collect the LB medium in the bottom for further shaking culture. Transfer E. Quantify E. coli using a multi-plate reader with an OD value of 600 nm.

[0087] 7. Corrosion resistance test of PHE hydrogel

[0088] The erosion of the PHE hydrogel was tested using a dilution method. Simply put, a cold PHE solution (2 mL) was placed in a scaling tube and allowed to gel completely in a constant temperature bath (37 °C). An excess of preheated PBS 7.4 (8 mL) was gently added to the PHE hydrogel, and the tube was then shaken at 12 rpm under constant temperature conditions (37 °C). The remaining volume of the hydrogel was recorded at different time intervals, and the erosion percentage was calculated using the following formula: Erosion percentage (%) = [1 - Vt / V0] × 100%, where Vt is the remaining hydrogel volume at time t and V0 is the initial volume.

[0089] 8. TNBS-induced ulcerative colitis in rats

[0090] Sprague-Dowley rats (male, 6-8 weeks old) were purchased from the Laboratory Animal Center of Wenzhou Medical University. All animals were housed in a specific pathogen-free barrier environment with food and water. Animal experiments were conducted according to the National Research Council's guidelines for the care and use of laboratory animals. Rats were fed normally for one week prior to the experiment to better acclimatize. Colitis was induced in rats using TNBS solution, following previous methods. The TNBS dose was 50 mg / kg, injected into the colon 6 cm from the anus via a polyethylene rubber catheter (3 mm in diameter). After instillation, the rats were inverted for one minute. As described in our previous studies, the establishment of the colitis rat model was successfully confirmed by weight loss, Disease Activity Index (DAI) scores, and mucosal ulceration.

[0091] 9. In vivo adhesion test of PHE hydrogel to mouse colon

[0092] To track PHE adhesion in the colon, bovine serum albumin (BSA) was labeled with fluorescent Cy7 propellant and encapsulated in a PHE hydrogel for IVIS imaging. First, the adhesion of the Cy7-labeled PHE hydrogel to the inflamed colon was evaluated in vitro. Simply put, the distal colon was excised from rats with TNBS-induced colitis, and the mucosa was exposed by inversion. Cy7-labeled PHE hydrogel (200 μL) was applied in vitro to the colonic mucosa and immersed in warm PBS solution (10 mL, 37 °C). At different time points, the colon was washed with fresh warm PBS and imaged using an IVIS imaging system (λex = 749 nm, λem = 767 nm). In vivo adhesion of the PHE hydrogel was also evaluated in healthy rats or rats with TNBS-induced colitis. Rats were fasted overnight, and Cy7-labeled PHE (500 μL) was injected into the colon 6 cm from the anus. Following injection, the animals were inverted for one minute. The fluorescence of rat abdominal tissues at different time intervals was imaged using an IVIS system. At the endpoint, all animals were sacrificed to obtain the colon. The fresh colon was frozen, sectioned, and stained with DAPI. Fluorescence within the colon was observed using a confocal laser scanning microscope. The in vivo adhesion of POL or DAP hydrogels as controls was tested.

[0093] 10. PHE-EK treatment of ulcerative colitis in rats

[0094] Rats with colitis were randomly divided into 5 groups: (1) TNBS group treated with PBS (TNBS group); (2) PHE hydrogel enema group (PHE group); (3) PHE-EGF group (2 μg / kg EGF per dose); (4) PHE-KPV group (10 μg / kg KPV per dose); (5) PHEEEK group (2 μg / kg EGF, 10 μg / kg KPV per dose). Healthy rats were also treated with PBS as a control (normal group). Each treatment was administered rectally, with 0.5 mL of test sample every 2 days for 14 consecutive days. The rats' body weight and DAI score were recorded throughout the process. Colonoscopy was also performed to image the colonic mucosa. Finally, the rats were sacrificed, and the colon and spleen were collected. The length of the colon was measured, and the weight of the spleen was recorded.

[0095] 11. Histological morphology

[0096] Fresh colon was fixed in 4% paraformaldehyde, then embedded in paraffin, and cut into 5 μm thick sections. After treatment with xylene and gradient ethanol solutions, the tissue sections were stained with H&E, Alixin Blue, periodic acid Schiff, or Massen trichrome. Additionally, deaffinity-treated tissue sections were treated with 5% BSA for 30 min, then incubated overnight at 4°C with primary antibodies in 1% BSA. Antibodies included anti-type I collagen (1:200, Abcam), anti-type III collagen (1:200, protein), anti-α-SMA (1:500, Abcam), and anti-mcp1 (1:200, Abcam). Sections were labeled using a Polink 1HRPDAB detection system (GBI, USA) and detected using a Nikon ECLPSE80i (Nikon, Japan).

[0097] 12. Pro-inflammatory cytokines

[0098] Immunohistochemical staining was used to detect pro-inflammatory cytokines (IL-6, TNF-α, IL-1β) and anti-inflammatory cytokines (IL-10). Briefly, de-affinized tissue sections were treated with 5% BSA for 30 min, then incubated with primary antibodies including anti-IL-1β (1:200, Abcam), anti-TNF-α (1:100, Santa Cruz), anti-IL-6 (1:200, affinity), and anti-IL-10 (1:200, AP) in 1% BSA overnight at 4°C. Sections were labeled using a Polink 1HRPDAB detection system (GBI, USA) and detected using a Nikon ECLPSE80i (Nikon, Japan).

[0099] 13. Tight junction proteins

[0100] Tight junction proteins (ZO1, Claudin-5) were detected using immunofluorescence staining. Primary antibodies included claudin-5 (1:100, AB clone) and ZO1 (1:200, ABclin clone). Secondary antibodies were donkey anti-rabbit IgG Alexa Fluor488 (1:1000, Abcam) or goat anti-mouse IgG Alexa Fluor488 (1:1000, Abcam). Sections were mounted and stained with DAPI (DAPI, 6-hook) and then observed using a confocal laser scanning microscope (Nikon, TiE & A1plus).

[0101] 14. Statistical Analysis

[0102] All data in this invention are expressed as mean ± standard error of the mean. To verify validity, three independent experiments were conducted, with at least three samples taken for each test for statistical analysis. Statistical analysis was performed using GraphPadPrism 8.0 software, and statistical comparisons were conducted using analysis of variance (ANOVA) or t-tests.

[0103] Results and Discussion

[0104] 1.1 Temperature Sensitivity of PHE Hydrogel

[0105] Polychlorosamine, also known as polyisocyanate Polyoxamine is the most common polymer for forming thermosensitive hydrogels. Among the variants of polyoxamine, P407 is widely used as a hydrogel-forming material due to its low toxicity and water solubility; however, P407 has the lowest hydrogel-forming concentration of 17% (w / v), and when applied to mucosal tissues, it generally cannot resist dilution with water. To overcome this drawback, dihydrocaffeic acid-modified doloxamine (DAP) was successfully synthesized. DAP not only retains the thermosensitivity of P407 but also has stronger mechanical strength and better tissue adhesion. Importantly, the DAP hydrogel can withstand dilution in PBS (10 mM) at pH 7.4 for 48 hours, significantly improving its anti-erosion properties. However, the DAP hydrogel is difficult to specifically adhere to the colon of colitis patients rather than to healthy patches. Based on the biological function of mucus, this invention combines temperature-sensitive DAP with EGCG, hyaluronic acid (HA), and growth factors (KPV and EGF) to design an in-situ hydrogel (PHE-EK). As shown in Figure 3A, both EGCG (1%, w / v) and HA (1%, w / v) readily dissolve in cold DAP solution (20%, w / v), resulting in a translucent, slightly brownish PHE solution. Furthermore, the addition of KPV, EGF, or both does not affect the formation and properties of the transparent PHE solution. The sol-gel transition of the PHE solution was observed in a 37°C constant-temperature bath, indicating its good temperature sensitivity. The gelation time of cold PHE was further determined using the tilting method. The PHE solution (1 mL) gelled rapidly within 10 s, comparable to the DAP solution (Figure 3B). However, the gelation time of the PHE solution was significantly shorter than that of the POL solution. The temperature sensitivity of the PHE solution was further demonstrated by temperature-dependent viscosity analysis (Figure 3C). The viscosity of POL increased slowly between 26 and 35°C, while the viscosity of DAP or PHE increased abruptly between 30 and 38°C. At the same temperature, the viscosity of PHE was slightly lower than that of DAP. Furthermore, the gelation temperature of PHE was further tested through rheological studies. As shown in Figure 3D, PHE gelled at 36°C, slightly higher than DAP (32°C).

[0106] 1.2 Viscoelastic behavior of PHE hydrogel

[0107] The viscoelastic properties of PHE hydrogels at 37°C were also measured using oscillation frequency scanning. The storage modulus (G') of the PHE hydrogel was higher than its loss modulus (G'), exhibiting elastic properties (Figure 3E). However, compared to DAP or POL hydrogels, the G' of the PHE hydrogel was slightly lower, indicating that the addition of HA or EGCG weakened the mechanical strength of the hydrogel. The mechanical strength (storage modulus, G') of the hydrogel largely depends on the crosslinking of the polymer. For POL hydrogels, the crosslinking within the hydrogel network is micelle assembly. In addition to micelle assembly crosslinking, the oxidation of the DAP polymer by phenolic DA also provides additional crosslinking for the DAP hydrogel. The mechanical strength of the PHE hydrogel is mainly determined by micelle assembly and oxidation-mediated crosslinking. Since the entanglement of HA with the DAP polymer hinders oxidation-mediated crosslinking, the addition of HA reduces the mechanical properties of the PHE hydrogel. The toughness of hydrogels has been widely used to characterize the ability of materials to resist fracture under mechanical loads. As shown in Figure 3F, the POL hydrogel exhibits relatively weak toughness with a fracture strain of 3.9%. Due to the self-crosslinking of DA, the DAP hydrogel, with a fracture strain of 24.7%, shows significantly enhanced toughness. In contrast, the PHE hydrogel shows an increased fracture staining rate of 77.8%, indicating that EGCG or HA further improves the toughness of the DAP hydrogel. The enhanced toughness of the PHE hydrogel may be due to the entanglement of HA and DAP polymers and the strong interaction between EGCG and DAP. These physical interactions between the components of PHE are supported by FT-IR spectroscopy. Similar phenomena have been observed in previous studies.30 The shear thinning effect of the PHE hydrogel is further confirmed by viscosity-shear curves (Figure 3G). Furthermore, the self-healing properties of the PHE hydrogel were also tested by the recovery rates at low strain, high strain (1%), or 100%. The PHE hydrogel exhibits a malignant state at high strain (G>G); however, at low strain, it can rapidly recover to the gel state (G>G) (Figure 3H). This indicates that the PHE hydrogel has good injectability and superior contact properties.

[0108] 1.3 Corrosion resistance of PHE hydrogel

[0109] To determine whether EGCG / HA affects the anti-erosion properties of DAP hydrogels, the in vitro erosion of PHE hydrogels was investigated, and the results are shown in Figure 3I. Similar to DAP hydrogels, PHE hydrogels exhibited slower erosion behavior than POL hydrogels. POL hydrogels were completely eroded within 36 hours, while PHE or DAP hydrogels retained over 70%, indicating that the anti-dilution properties of PHE hydrogels were not affected. The dense three-dimensional mucus network is crucial for preventing pathogens from entering the colonic lumen via epithelial or immune cells. SEM images of PHE hydrogels showed a denser three-dimensional network compared to DAP or POL hydrogels (Figure 3J). The average pore size of PHE hydrogels was ca. 30 μm, smaller than that of POL (ca. 80 μm) or DAP (60 μm). The smaller network size of the simulated mucus hydrogels is beneficial for preventing the invasion of intestinal microbiota without affecting the diffusion of nutrients such as short-chain fatty acids (DCFAs). The dense network of PHE hydrogels may be related to the physical entanglement of HA or the interaction between EGCG and DAP, as confirmed by the rheological results above.

[0110] 1.4 In vitro adhesion test of PHE hydrogel

[0111] The tissue adhesion of PHE hydrogel to rat skin was first evaluated, and the results are shown in Figures 4A, 4B, 4C, and 4D. Due to the phenolic da-mediated interaction with tissue, DAP hydrogel exhibited stronger tissue adhesion. Compared to DAP hydrogel, PHE hydrogel showed significantly higher adhesion strength (1421±96 mN) and adhesion force (22±2 J·m⁻²) than POL hydrogel. EGCG, a polyphenolic additive, has been reported to enhance the tissue adhesion properties of PLGA-PEG-PLGA hydrogels in previous studies. In this invention, the polysaccharide of HA may counteract the enhanced adhesion of EGCG in PHE hydrogel. Antimicrobial peptides, transferrin, and bactericidal / permeability-enhancing proteins pathologically accumulate on the mucosa of the inflamed colon, resulting in an excess of positive charge. HA not only has a net negative charge on its molecular backbone but also targets CD44, a transmembrane glycoprotein overexpressed on epithelial cells and macrophages of the inflamed intestine.

[0112] To determine whether PHE hydrogel can target inflamed colon via electrostatic effects, three PVA film substrates (neutral, positive, and negatively charged) were prepared to test the adhesion force and adhesion performance of PHE hydrogel. The results are shown in Figures 4E, 4F, 4G, 4H, and 4I. The adhesion force and adhesion work of PHE hydrogel to positively charged substrates reached 3206.47±569.54 mN and 89.80±17.86 J·m², respectively, significantly higher than those of DAP and POL hydrogels. Furthermore, the adhesion force and adhesion work of PHE hydrogel to neutrally and negatively charged substrates were not significantly different from those of DAP or POL hydrogels. In contrast, DAP and POL hydrogels showed no significant difference in adhesion force and adhesion work to these substrates. These results indicate that PHE hydrogel can specifically adhere to positively charged surfaces.

[0113] 1.5 PHE's in vitro antibacterial activity

[0114] E. coli was used as an opportunistic pathogen model. As shown in Figures 5A and 5B, the colony count in the POL group was comparable to that in the PBS control group. Treatment with DAP or PHE significantly reduced colony counts, demonstrating strong antibacterial activity. PHE achieved a bacterial kill efficiency of 99.9%, higher than DAP. The antibacterial effect of DAP may be due to the catechins at the DA terminal. Similar antibacterial phenomena were observed in catechol-derived films. This explains why PHE exhibits stronger antibacterial activity than DAP. Optical density (OD600) was measured after culturing E. coli. The results are shown in Figure 5C. The OD600 values ​​for the DAP and PHE groups were 0.16 ± 0.03 and 0.12 ± 0.02, respectively, significantly lower than those for PBS or POL. However, there was no statistically significant difference in OD600 values ​​between the DAP and PHE groups. Further migration of E. coli was mediated using a transporous device on the PHE hydrogel (Figure 5D). The number of E. coli is shown in Figure 5E. When the cavity was enclosed by PBS, E. coli migrated downwards. The bacterial migration rates in both the POL and DAP groups were significantly reduced. However, the OD values ​​in the upper and lower cavities were comparable in both groups, indicating limited resistance to bacterial invasion. Interestingly, very few bacterial colonies migrated in the PHE group. The OD value of the PHE hydrogel in the lower cavity was three times that in the upper cavity, demonstrating that the PHE hydrogel effectively inhibited bacterial invasion.

[0115] 1.6 PHE hydrogel in vivo adhesion on inflamed colon

[0116] First, in vitro imaging was used to evaluate the adhesion of PHE hydrogel to the inflamed colon (Figure 6A). The results are shown in Figures 6B and 6E. The fluorescence of POL or DAP decreased significantly within 2 hours, indicating their poor retention on the colitis colonic mucosa. In contrast, PHE hydrogel did not show significant fluorescence decay at the same time point. Furthermore, even after 12 hours of washing, more than 60% of PHE was retained on the colitis colon (Figure 6E), indicating its strong adhesion. The strong adhesion of PHE is due to the following reasons: First, the hydrogen bonding interaction between HA, DAP, and EGCG gives PHE anti-erosion properties exceeding those of water; second, the phenol-mediated interaction between EGCG and the mucosa also involves tissue adhesion; third, the electrostatic interaction between HA and the inflamed colonic mucosa may also enhance the specific adhesion of PHE to the colitis colonic mucosa. In vivo abdominal imaging of PHE hydrogel in healthy rats or TNBS-induced colitis rats also confirmed the specific adhesion of PHE hydrogel to the inflamed colon. As shown in Figures 6C and 6F, intestinal fluorescence was detected in colitis rats after administration. However, the intestinal fluorescence spectra of these groups changed over time. Intestinal fluorescence in rats treated with POL or DAP decayed rapidly over time, with only weak fluorescence distributed in the intestinal region at 8 hours. In contrast, PHE-treated rats showed a slower fluorescence decay curve, with strong intestinal fluorescence still visible even at 8 hours. Furthermore, at each time point, PHE showed more pronounced fluorescence in TNBS-treated rats, indicating that the PHE hydrogel could specifically adhere to the inflamed colon. There was no statistically significant difference in fluorescence intensity between the DAP and POL groups. This suggests that neither DAP nor POL showed specific enrichment on the inflamed colon. All animals were sacrificed 8 hours after administration, and colonic tissue was collected, then sectioned for in situ imaging. Similar results were obtained, as shown in Figures 6D and 6G. The retention of PHE hydrogel on the colonic mucosa in colitis rats was higher than in normal rats. In conclusion, PHE has the ability to target inflamed sites and prolong the retention time at these sites.

[0117] 1.7 PHE-EK can alleviate TNBS-induced colitis in rats.

[0118] In vitro cell experiments confirmed the good biocompatibility of PHE with Caco-2. EGF, a potent mitotic peptide, has been shown to be beneficial for neonatal necrotizing enterocolitis. The KPV tripeptide (Lys-Pro-Val) derived from the C-terminal sequence of α-melanocyte-stimulating hormone (α-MSH) has been shown to be effective against colitis in mice. Mucus-inducing hydrogels with regenerative activity (PHE-EGF, PHE-KPV, or PHE-EK) were prepared by adding KPV or EGF, or both, to PHE. The bioactivity of this 14EGF or KPV in the PHE hydrogel was well preserved, as confirmed by cell proliferation assays. The alleviating effect of PHE-EK on TNBS-induced colitis in rats was evaluated using the experimental protocol shown in Figure 7A. The TNBS group (untreated) initially showed significant weight loss and colitis-related symptoms (diarrhea or fecal bleeding), with a higher DAI score of 5 on day 1 after TNBS treatment (Figures 7B and 7C). Compared with the TNBS group, TNBS-induced colitis rats showed significantly improved weight loss and DAI scores at 10 years of age in the PHE-EGF, PHE-KPV, or PHE-EK treatment groups (Figures 7B and 7C). Furthermore, among these treatments, PHE-EK showed the most significant improvement in colitis-related symptoms. At the endpoint, colons of all rats were collected for observation, and colon length was counted. As shown in Figures 7D and 7E, these treatments (PHE-EGF, PHE-KPV, or PHE-EK) significantly inhibited colonic shortening in TNBS-induced colitis rats. Colon length in these groups recovered to levels comparable to healthy rats. There was no statistically significant difference in colon length among the PHE-EGF, PHE-KPV, and PHE-EK groups. However, the spleen weight of colitis rats treated with PHE-EK or PHE-KPV was significantly smaller than that in the PHE-EGF treatment group (Figure 7F). The spleen is an immune-related organ and is often used as an important indicator of the severity of inflammation. These results suggest that KPV has strong anti-inflammatory activity. In dss-colitis mice, KPV showed stronger anti-inflammatory effects than α-MSH. Furthermore, during treatment, colonic bleeding and ulceration were observed in real-time using endoscopic imaging, as shown in Figure 7G. On day 7, ulcerated mucosal patches with visible bleeding points appeared in the TNBS group. After two weeks of treatment with PHE, PHE-EGF, or PHE-KPV, the ulcer area decreased, and the colonic mucosa partially recovered. However, hematomas were still visible in the colonic mucosa of these groups. In addition, thin and fragile colonic mucosa remained in these groups. Colitis rats treated with PHE-EGF or PHE-KPV showed severe colonic stenosis and moderate edema. However, no mucosal edema or bleeding was observed in PHE-EK treatment (Figure 7G), indicating the most significant improvement in mucosal healing.H&E staining revealed inflammatory cell infiltration in the colonic mucosa of the TNBS group, with severe collapse of epithelial cells and crypts (Fig. 7H). PHE-EK treatment rescued rats from TNBS damage by reducing inflammatory cell infiltration and restoring crypts and mucosal epithelium. Furthermore, PAS and Masson staining showed that after PHE-EK treatment, epithelial goblet cells were well-aligned, and the remodeling of submucosal fibrosis-related collagen was significantly inhibited. The repairing effect of PHE-EK may be related to EGF. Intravenous injection of exogenous EGF in patients with gastric ulcers has been shown to effectively induce epithelial-mucosal barrier repair. In addition, oral administration of high doses of recombinant human EGF to patients with gastrointestinal ulcers also accelerated ulcer healing. Furthermore, rectal administration of rhEGF (5 μg) in a degraded and modified gelatin carrier solution has shown a positive effect on clinical remission scores in patients with active left-sided ulcerative colitis. In summary, these findings suggest that PHE-EK mimics healthy mucus and may repair the mucosal barrier.

[0119] 1.8 PHE-EK reduced the expression of pro-inflammatory cytokines.

[0120] TNBS-induced colitis involves a complex cascade of reactions, including the recruitment and subsequent infiltration of inflammation-associated immune cells, such as CD3+ infiltrating T cells and CD68+ macrophages, which further secrete excessive amounts of pro-inflammatory cytokines (e.g., IL1β, IL-6, and TNFα).43. Immunohistochemical staining for IL1β, IL-6, and TNFα is shown in Figure 8A. Indeed, high expression of IL-6, TNF-α, or IL1β was observed in the lamina propria or epithelial cells of the colon in rats with TNBS-induced colitis. As expected, PHE-EGF had a slight inhibitory effect on IL1β, IL-6, or TNF-α, indicating limited anti-inflammatory activity. However, the expression of IL-6 or TNFα was significantly suppressed after treatment with PHE-EK or PHE-KPV. Furthermore, compared with the TNBS group, both PHE-KPV and PHE-EK upregulated the anti-inflammatory cytokine (IL10). These results suggest that PHE-KPV or PHE-EK treatment has a strong anti-inflammatory response. However, compared to PHE-KPV, PHE-EK showed a more significant decrease in IL-6 or TNF-α expression (Figure 8B). The most pronounced inflammatory relief in PHE-EK was due to the synergistic effect of KPV and EGF. Similar inflammatory relief results have been observed in previous studies.

[0121] 1.9 PHE-EK upregulated the expression of tight junction proteins.

[0122] The intestinal mucosal barrier, composed of mucus, epithelial cells, and immune cells in the lamina propria, is a crucial defense against intestinal bacteria and food-derived antigens. Tight junctions (TJs) between epithelial cells play a key role in maintaining the integrity of the intestinal mucosal barrier; damage to the mucosal barrier is highly correlated with the pathogenesis of colitis. Repairing the intestinal mucosal barrier and maintaining its dynamic homeostasis are critical for the treatment of ulcerative colitis (UC). Tight junctions are multi-protein complexes composed of transmembrane proteins, peripheral membrane (scaffold) proteins, and regulatory kinases, and they are the main determinants of mucosal permeability. ZO1 and Claudin-5 are two markers of tight junction proteins. Immunofluorescence staining was used to detect the expression of ZO1 and Claudin-5, and the results are shown in Figure 9A. After TNBS treatment, most of the epithelial cells collapsed, and TJs (ZO1 and Claudin-5) were severely damaged. Similarly, epithelial cell recovery was poor after PHE-KPV, and TJ expression did not improve. In contrast, both ZO1 and Claudin-5 expression were significantly upregulated after treatment with PHE-EGF or PHE-EK. Particularly during PHE-EK treatment, epithelial cells were well-aligned, and transfer junctions (TJs) were most prominent (Figure 9B). These results suggest that the repair of the intestinal mucosal barrier may be primarily attributed to EGF activity. However, the anti-inflammatory effect of KPV may also promote intestinal mucosal barrier repair, leading to a more pronounced upregulation of TJs during PHE-EK treatment.

[0123] 1.10 PHE-EK remodeled the extracellular matrix

[0124] Fibrosis is defined as the excessive accumulation of collagen-rich extracellular matrix (ECM) and is a common complication of various chronic diseases. Colonic fibrosis is a major challenge in the treatment of colitis, leading to irreversible colonic stenosis. Collagen-i is a major component of the ECM, possessing a fractal microstructure and rigid matrix properties; while collagen-iii represents scarless healing of damaged tissue. Activated mesenchymal cells are highly correlated with fibrotic remodeling and participate in ECM synthesis; α-SMA is a typical marker of activated myofibroblasts. To confirm whether mucosal barrier repair is accompanied by fibrotic remodeling of the extracellular matrix, we further examined α-SMA, collagen-i, and type-iii collagen. As shown in Figure 10, the TNBS group showed abundant production of type-i and type-iii collagen in the lamina propria and muscularis mucosae. Furthermore, the ratio of type-iii / i collagen was lower in the TNBS group. Additionally, a large amount of α-SMA was expressed in the lamina propria of the TNBS group. These results are consistent with the colonic swelling observed in this group, suggesting the presence of pathological fibrosis progression. EGF possesses activities that promote cell proliferation, differentiation, and migration, which are associated with the fibrotic process. As expected, PHE-EGF treatment did not significantly inhibit collagen-i deposition or α-SMA expression, and significant fibrosis was observed in the lamina propria. In contrast, PHE-EK treatment significantly reduced the expression and deposition of collagen-i and α-SMA in the lamina propria and muscularis mucosae (Figs. 10A and 10B). Compared with TNBS, PHE-EGF, or PHE-KPV, the collagen-iii / collagen-i ratio was significantly increased in the PHE-EK group. Monocyte chemoattractant protein 1 (MCP1) is thought to play an important role in the induction of colonic fibrosis in mice. It was overexpressed in the colonic epithelium of the PHE-EGF group and significantly downregulated in the colonic tissue, consistent with collagen deposition after PHE-EK treatment. These results indicate that PHE-EK requires KPV in combination with EGF to promote non-fibrotic ECM remodeling in TNBS-induced colitis rats.

[0125] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A mucus-inducing hydrogel with regenerative activity, characterized in that, The regenerative mucus-inducing hydrogel is composed of dihydrocaffeic acid-modified poloxamine, hyaluronic acid, epigallocatechin-3-gallate, and bioactive factors, namely KPV tripeptide and epidermal growth factor.

2. The mucus-activated hydrogel with regenerative activity as described in claim 1, characterized in that, PHE-EK is a flowable liquid at room temperature and gels within 10 seconds at body temperature.

3. The mucus-activated hydrogel with regenerative activity as described in claim 1, characterized in that, The strain of the PHE-EK hydrogel is 77.8%.

4. The mucus-activated hydrogel with regenerative activity as described in claim 1, characterized in that, PHE-EK hydrogels specifically adhere to the inflamed colon through electrostatic interactions.

5. The mucus-activated hydrogel with regenerative activity as described in claim 1, characterized in that, After PHE-EK treatment, epithelial cells were well-aligned and tight junction proteins were significantly upregulated. PHE-EK also promoted non-fibrotic remodeling of the extracellular matrix of colon cells by upregulating the collagen-iii / collagen-i ratio and reducing the expression of MCP1 and α-SMA.

6. The method for preparing a regenerative mucus-activated hydrogel according to any one of claims 1 to 5, characterized in that, The method for preparing the regenerative mucus-inducing hydrogel includes: integrating temperature-sensitive dihydrocaffeic acid-modified doloxamine (DAP) with antibacterial components EGCG and hyaluronic acid (HA) to prepare in-situ hydrogel PHE; adding KPV and EGF to growth factors to form PHE hydrogel, generating mucus-inducing hydrogel PHE-EK.

7. The method for preparing the regenerative mucus-activated hydrogel as described in claim 6, characterized in that, The PHE solution was prepared using a gradient dissolution method, specifically including the following steps: Step 1, dissolving 1.0 g of HA powder in 100 mL of distilled water; Step 2, dissolving 20.0 g of DAP powder in a cold HA solution at 4 °C and stirring gently; Step 3, further dissolving 1.0 g of EGCG to obtain a malignant PHE solution; Step 4, directly dissolving EGF, KPV, or both in the cold PHE solution to prepare a PHE-EK solution.

8. The application of a mucus-stimulating hydrogel with regenerative activity as described in any one of claims 1 to 5 in the treatment of ulcerative colitis.

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