Collagen hydrogel, preparation method thereof and application of collagen hydrogel in delivery of hangover-alleviating active enzyme

Abstract

The invention relates to collagen hydrogel, a preparation method thereof and application of the collagen hydrogel in delivery of hangover-alleviating active enzyme. According to the collagen hydrogel provided by the invention, the collagen-based hydrogel is combined with the micro-scale grid and the artificial villus structure, so that the effects of prolonging the retention time in the stomach, enhancing the mechanical toughness and improving the compressive strength are achieved under physiological conditions, and the long-standing problem of balancing between the mechanical stability and the catalytic efficiency is solved; meanwhile, the excellent biocompatibility is realized. Taking hangover alleviating application as an example, the acetaldehyde removal efficiency of the system is improved by 30 times in a simulated stomach environment by fixing a double-enzyme cascade system of alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH), and good quick hangover alleviating application potential is shown.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, in particular to a collagen hydrogel, a preparation method thereof and application thereof in oral delivery of bioactive enzymes such as ethanol dehydrogenase and acetaldehyde dehydrogenase. BACKGROUND

[0002] Alcohol consumption is deeply rooted in global culture and social practice, but the strong reactivity and cytotoxicity of acetaldehyde, an intermediate metabolite, pose a serious health threat to the human body. In particular, about 40% of East Asian populations have ALDH2 gene polymorphism, which reduces the ability to clear acetaldehyde, thereby increasing the risk of gastrointestinal diseases and alcohol-related cancers.

[0003] The ethanol metabolism process generates acetaldehyde, a cytotoxic intermediate product, which accumulates and poses a serious threat to health, especially for individuals with defective acetaldehyde dehydrogenase (ALDH) activity. An ideal detoxification strategy needs to meet the following key conditions: (1) have a deformable or inflatable structure to prolong the residence time in the stomach; (2) have sufficient mechanical strength to withstand gastrointestinal peristalsis; (3) maintain sustained biocatalytic activity under physiological conditions; (4) have good biocompatibility to ensure oral safety. Overcoming the above challenges will promote the clinical translation of enzyme therapy in efficient acetaldehyde clearance and other detoxification applications.

[0004] Traditional oral enzyme supplementation faces many problems, such as rapid clearance in the stomach, enzyme inactivation under strong acidic conditions, and low substrate-enzyme contact efficiency. In recent years, although progress has been made in hydrogel-based drug delivery systems, there are still core problems: enhancing mechanical stability to prolong gastric residence time limits enzyme contact with substrates, and improving enzyme activity sacrifices gel structure strength. This is because traditional hydrogels are difficult to meet three key design parameters at the same time: (1) good gastric adhesion; (2) stable enzyme protection ability; (3) high substrate diffusion capacity, which is an important obstacle to the clinical application of enzyme therapy.

[0005] Therefore, how to construct a hydrogel system with strong gastric residence time, mechanical toughness and compressive strength has become a technical problem to be solved. SUMMARY

[0006] To solve the above technical problems, the present application provides a collagen hydrogel, a preparation method thereof and application thereof, which realizes long gastric residence time, enhanced mechanical toughness and excellent compressive strength under physiological conditions, and solves the long-standing trade-off between mechanical stability and catalytic efficiency.

[0007] To achieve this purpose, the present application adopts the following technical solutions:

[0008] In a first aspect, the present application provides a preparation method of a collagen hydrogel, the preparation method comprising mixing collagen, an acrylamide solution, a bisacrylamide solution and a calcium chloride solution, ultrasonicating, and then adding an oxidant and a reducing agent and standing.

[0009] In the present application, a collagen-based hydrogel framework is constructed, which has a villus-like microstructure on the surface, can significantly increase the specific surface area and thus improve the enzyme loading capacity, and the flexible villi can physically interlock with the gastric mucosa to enhance adhesion and prolong residence time. The biomimetic structure not only improves the interfacial adhesion, but also promotes the dynamic diffusion of substrates, significantly improving the catalytic efficiency. In the raw materials of the hydrogel prepared in the present application, a bisacrylamide solution and a calcium chloride solution are also introduced. The bisacrylamide solution, as one of the main polymerization monomers, can increase the mechanical strength of the gel, increase the number of crosslinking points, and make the three-dimensional grid more stable. At the same time, it can regulate the pore size and swelling performance, improve the structural stability, and the calcium chloride solution can physically crosslink with polymers and biological macromolecules with functional groups such as carboxyl (–COO⁻), hydroxyl (–OH), and amide (–CONH2), forming a double network structure (covalent crosslinking + ionic crosslinking) to improve the mechanical properties and toughness. At the same time, calcium ions play an important role in the extracellular matrix and signal transduction, and can improve the biological adaptability in the collagen hydrogel system.

[0010] Preferably, the collagen, the acrylamide solution, the bisacrylamide solution and the calcium chloride solution are in a ratio of (0.1-0.5) g:(0.8-1.2) mL:(50-150) μL:100 μL. The (0.1-0.5) g may, for example, be 0.1 g, 0.2 g, 0.3 g, 0.4 g or 0.5 g, etc. The (0.8-1.2) mL may, for example, be 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL or 1.2 mL, etc. The (50-150) μL may, for example, be 50 μL, 60 μL, 70 μL, 80 μL, 90 μL, 100 μL, 110 μL, 120 μL, 130 μL, 140 μL or 150 μL, etc.

[0011] Preferably, the mass concentration of acrylamide in the acrylamide solution is 30%-50%. For example, it may be 30%, 35%, 40%, 45% or 50%, etc.

[0012] Preferably, the mass concentration of bisacrylamide in the bisacrylamide solution is 10%-30%. For example, it may be 10%, 15%, 20%, 25% or 30%, etc.

[0013] Preferably, the molar concentration of calcium chloride in the calcium chloride solution is 2-4 M. For example, it can be 2 M, 2.5 M, 3 M, 3.5 M or 4 M, etc.

[0014] Preferably, the oxidizing agent comprises any one or a combination of at least two of ammonium persulfate, potassium persulfate or 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropionphenone;

[0015] Preferably, the molar concentration of the oxidizing agent is 1-3 M. For example, it can be 1 M, 1.5 M, 2 M, 2.5 M or 3 M, etc.

[0016] Preferably, the ratio of the oxidizing agent to collagen is (60-80) μL:(0.1-0.5) g. The (60-80) μL, for example, can be 60 μL, 65 μL, 70 μL, 75 μL or 80 μL, etc. The (0.1-0.5) g, for example, can be 0.1 g, 0.2 g, 0.3 g, 0.4 g or 0.5 g, etc.

[0017] Preferably, the reducing agent comprises any one or a combination of at least two of tetramethylethylenediamine, N,N-dimethyl-p-toluidine or piperazine;

[0018] Preferably, the ratio of the reducing agent to collagen is (5-15) μL:(0.1-0.5) g. The (5-15) μL, for example, can be 5 μL, 6 μL, 7 μL, 8 μL, 9 μL, 10 μL, 11 μL, 12 μL, 13 μL, 14 μL or 15 μL, etc. The (0.1-0.5) g, for example, can be 0.1 g, 0.2 g, 0.3 g, 0.4 g or 0.5 g, etc.

[0019] Preferably, the temperature for the standing is 0-8℃ and the time is 8-20 h. The 0-8℃, for example, can be 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, 6℃, 7℃ or 8℃, etc. The 8-20 h, for example, can be 8 h, 10 h, 12 h, 14 h, 16 h, 18 h or 20 h, etc.

[0020] In a second aspect, the present application provides a collagen hydrogel prepared by the method according to the first aspect.

[0021] In a third aspect, the present application provides a use of the collagen hydrogel according to the second aspect in the preparation of a drug delivery system.

[0022] In a fourth aspect, the present application provides an oral delivery system with alcoholism treatment function, which comprises the collagen hydrogel of the second aspect and an active enzyme.

[0023] In the present application, the constructed collagen hydrogel framework has good buffering effect on acidic environment, so that the enzyme remains active in the stomach, effectively preventing enzyme inactivation. In a simulated gastric environment, the acetaldehyde removal efficiency of the system is improved by 3020%, showing good treatment potential.

[0024] Preferably, the active enzyme comprises ethanol dehydrogenase and / or acetaldehyde dehydrogenase.

[0025] Preferably, the loading amount of the alcoholism treatment active enzyme is 1%-5% of the mass of the collagen hydrogel. For example, it can be 1%, 2%, 3%, 4% or 5%, etc.

[0026] In a fifth aspect, the present application provides a use of the oral delivery system with alcoholism treatment function according to the fourth aspect in the preparation of an alcoholism treatment drug.

[0027] The application of the present application realizes rapid alcoholism treatment taking alcohol metabolism enzyme as an example, and is suitable for the immobilization and delivery of other biological enzymes.

[0028] Compared with the prior art, the present application has at least the following beneficial effects:

[0029] The present application constructs a collagen-based hydrogel framework, and introduces a micro-scale grid and an artificial villus structure to fix a dual-enzyme system of ethanol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH). Inspired by the natural villi of the gastrointestinal tract, the surface of the hydrogel has a villus-like microstructure, which can significantly increase the specific surface area and improve the enzyme loading capacity. At the same time, the flexible villi can physically interlock with the gastric mucosa, enhance adhesion, and prolong residence time. The biomimetic structure not only improves the interfacial adhesion, but also promotes the dynamic diffusion of the substrate, significantly improving the catalytic efficiency. In addition, the collagen hydrogel framework has good buffering effect on acidic environment, so that the enzyme remains active in the stomach, effectively preventing enzyme inactivation. In a simulated gastric environment, the acetaldehyde removal efficiency of the system is improved by 3020%, showing good treatment potential. The present application proposes a multi-scale material engineering strategy to maintain mechanical stability while improving enzyme catalytic efficiency, providing a new direction for gastrointestinal enzyme therapy. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1Figure 1. Schematic diagram of the construction principle and mechanism of ADH / ALDH@VH hydrogel. a, Schematic diagram of the design and delivery process of ADH / ALDH@VH. b, Schematic diagram of the accumulation of acetaldehyde in the alcohol metabolism pathway in the absence of ALDH, inducing toxic reactions. c, Schematic diagram of the rapid metabolism of acetaldehyde to acetic acid by exogenous ALDH in the stomach after the use of ADH / ALDH@VH, significantly reducing toxic reactions. d, Schematic diagram of the hydrophobic interaction between ALDH and the hydrophobic groups in the collagen hydrogel scaffold, enhancing the stability of its embedding in the hydrogel. e, Schematic diagram of the hydrogen bonding between the functional groups on the surface of the ALDH molecule (such as amino, hydroxyl, and carboxyl groups) and the amide or carboxyl groups in the hydrogel.

[0031] Figure 2 Figure 2. Schematic diagram of the mechanical strength test of ADH / ALDH@VH hydrogel.

[0032] Figure 3 Figure 3. Schematic diagram of the long-term stability of ADH / ALDH@VH hydrogel. a, In the free state, ALDH loses the ability to metabolize acetaldehyde after long-term storage. b, After loading into the hydrogel, acetaldehyde can still be metabolized to acetic acid after long-term storage.

[0033] Figure 4 Figure 4. Schematic diagram of the water absorption performance test of the hydrogel.

[0034] Figure 5 Figure 5. Performance test diagram of the hydrogel. a, Morphological changes of ADH / ALDH@VH under different deformation states. b, Cycle number-stress curve of 600 stretching-release cycle tests at room temperature. c, Swelling rate-compression stress time curve. d, Cycle number-stress curve of 550 compression-release cycles at the maximum swelling state. e, Morphological changes before and after swelling. f, Adhesion strength changes of different biological substrates in lap shear tests. g, Skin condition before and after natural removal of ADH / ALDH@VH attached to human skin.

[0035] Figure 6 Figure 6. Schematic diagram of the mechanical property optimization of the hydrogel. a, Tensile stress-strain curve under different collagen concentrations. b, Compression stress-strain curve under different collagen concentrations.

[0036] Figure 7 Figure 7. Schematic diagram of the mechanical property optimization of the hydrogel loaded with alcohol dehydrogenase. a, Tensile stress-strain curve of the hydrogel loaded with alcohol dehydrogenase. b, Compression stress-strain curve of the hydrogel loaded with alcohol dehydrogenase.

[0037] Figure 8 Figure 8. Comparison chart of the maximum compressive strength and fracture strain of the hydrogel before and after loading alcohol dehydrogenase.

[0038] Figure 9 Swelling properties of hydrogels at different collagen concentrations.

[0039] Figure 10 Hydrogel morphology changes over time when a compression force is applied to ADH / ALDH@VH hydrogel with a weight.

[0040] Figure 11 Schematic diagram of the experimental device for lap shear adhesive test.

[0041] Figure 12 Biocompatibility detection diagram, wherein a diagram is a staining diagram of GES-1 cells in a blank control group, b diagram is a result diagram of staining GES-1 cells with ADH / ALDH@VH extraction solution, c diagram is a cell survival rate result diagram, d diagram is an apoptosis result diagram of the control group cells, and e diagram is an apoptosis result diagram of the experimental group cells.

[0042] Figure 13 Morphological structure detection diagram of hydrogel, wherein a diagram is a comparison diagram of grid-shaped hydrogel structures with different morphologies and thicknesses, b diagram is a comparison diagram of relative enzyme activities of hydrogel samples with different morphologies and thicknesses, c diagram is a comparison of enzyme kinetic parameters of ADH / ALDH@VH and free ALDH, d diagram is an actual appearance image of ADH / ALDH@VH, e diagram is a close-up of artificial villus microstructure microscope of simulated gastric mucosa, f diagram is a diagram of ADH / ALDH@VH placed in a pig stomach model, respectively, after 7 minutes, g diagram is a diagram of ADH / ALDH@VH placed in a pig stomach model, respectively, after 30 minutes, and h diagram is a comparison diagram of acetaldehyde degradation efficiency of ADH / ALDH@VH group and untreated group in a simulated environment. DETAILED DESCRIPTION

[0043] The technical solutions of the present application will be further described below in combination with the drawings and through specific embodiments. However, the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0044] Example 1

[0045] Preparation of collagen hydrogel in this example

[0046] Collagen solution: 0.8575 g collagen was added to 2.75 mL of 40 wt% acrylamide, 300 μL of 0.2 wt% methylene bisacrylamide solution, 300 μL of 3 M CaCl2solution, and ultrasonicated for 30 min to obtain a homogeneous precursor solution. After adding 70 μL of 0.2 M ammonium persulfate and 10 μL of tetramethylethylenediamine, the solution was immediately injected into a mold (30 mm x 10 mm x 1.5 mm in size) and left to gel at 4 °C overnight to obtain a Col hydrogel.

[0047] ADH and ALDH were dissolved in the Col hydrogel solution, both at a 5% ratio, and added to the mold to obtain ADH / ALDH@VH.

[0048] Example 2

[0049] This example analyzes the hydrogel structure

[0050] As shown in FIG. a of Figure 1 The present application constructs an oral delivery system for loading alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) to improve the metabolic efficiency of acetaldehyde in the stomach. The system uses a water-sensitive expandable hydrogel matrix to achieve local and efficient delivery of enzymes, thereby promoting the rapid detoxification of acetaldehyde in the stomach. To increase the contact interface between the loaded enzymes (ADH / ALDH) and the gastric contents, the hydrogel is designed as a grid structure by Example 1. To further enhance its residence in the stomach and catalytic performance, artificial microvilli are constructed on the surface of the grid structure by Example 1. Under mild heating conditions, the hydrogel can be dehydrated into a dense solid, suitable for oral administration; after ingestion, the system can quickly rehydrate in gastric juice, restoring its original three-dimensional structure. At the same time, the artificial microvilli unfold and firmly adhere to the gastric mucosa, enhancing contact with the tissue and prolonging residence time, thereby improving the bioavailability and sustained activity of the enzymes.

[0051] As shown in the enlarged schematic diagram in FIG. a of Figure 1 The catalytic mechanism of the system is shown in the enlarged schematic diagram in FIG. a: the dual enzymes immobilized in the hydrogel rapidly catalyze the oxidation of ethanol to acetaldehyde (with a catalytic efficiency as high as 121 μM / mg / min), and then further oxidize acetaldehyde to acetic acid, thereby significantly improving the clearance efficiency of acetaldehyde, a toxic intermediate product. This pathway is the main route for acetaldehyde detoxification in the body, helping to reduce its accumulation in the stomach and alleviate the toxic effects of alcohol.

[0052] Figure 1 As shown in FIG. b and FIG. c of 1, the therapeutic mechanism of the exogenous ALDH system is further illustrated. As shown in Figure 1As shown in Figure b, ethanol is first oxidized to acetaldehyde under ADH catalysis, and then converted to acetic acid under ALDH catalysis. However, in individuals with impaired ALDH activity, acetaldehyde can accumulate in the body, leading to acute adverse reactions such as dizziness, flushing, tachycardia, and stomach discomfort. In contrast, as... Figure 1 Figure c shows that oral administration of ADH / ALDH@VH can significantly increase ALDH activity in the gastrointestinal tract and accelerate acetaldehyde degradation, thereby effectively alleviating alcohol-related symptoms.

[0053] To ensure stable encapsulation and controlled release of the enzyme within the hydrogel, we further investigated the molecular interactions between the enzyme and the collagen hydrogel. For example... Figure 1 As shown in Figures d and e, the hydrogel forms a three-dimensional cross-linked polymer network that effectively encapsulates enzyme molecules. Enzymes are primarily immobilized non-covalently through hydrophobic interactions and hydrogen bonding. Specifically, the hydrophobic residues on the enzyme surface interact with the hydrophobic regions within the hydrogel, helping to stabilize the enzyme conformation and prevent aggregation in an aqueous environment. Simultaneously, hydrogen bonds form between the functional groups (–NH3⁺, –OH, –COO⁻) on the enzyme molecule and the groups (–NH3⁺, –COO⁻) in the hydrogel backbone, thereby enhancing the enzyme's spatial anchoring effect and maintaining its structural stability under physiological temperature and pH conditions.

[0054] Test Example 3

[0055] This test case examines the mechanical strength of the hydrogel.

[0056] ADH / ALDH@VH hydrogels exhibit good mechanical strength, with a compressive strength as high as 288 kPa at 50% strain. Furthermore, as... Figure 2 As shown, it exhibits excellent biocompatibility in gastric tissue and superior time stability compared to free enzymes, such as... Figure 3 As shown, this is beneficial for long-term storage and practical application.

[0057] The physical and mechanical properties of the hydrogel were systematically tested. The results are as follows: Figure 4 As shown, the hydrogel has a water content as high as 80% and exhibits excellent flexibility, such as... Figure 5 As shown in Figure a, it can easily withstand large deformations such as stretching, torsion, and bending. To optimize its mechanical properties, we studied the effect of collagen concentration on the hydrogel properties, setting the collagen concentration to 0%-20% and conducting tensile tests using a universal tensile testing machine. Figure 6 As shown, with increasing collagen concentration, the tensile fracture strain, maximum tensile strength, and compressive strength of the hydrogel all significantly increased. Considering all factors, a collagen concentration of 20% was selected as the optimal substrate for enzyme encapsulation. Figure 7As shown, embedding ADH and ALDH into the hydrogel has almost no effect on its mechanical properties, indicating that ADH and ALDH are mainly embedded in the hydrogel in a non-covalent manner without destroying the integrity of its network structure.

[0058] To test the mechanical durability of the hydrogel under dynamic stress, we conducted a tensile-relaxation cycle test using a universal tensile testing machine. Figure 5 As shown in Figure b, the maximum tensile stress of the hydrogel did not change after 600 cycles, proving that it has excellent fatigue resistance and stability under repeated deformation.

[0059] Further testing was conducted on the expansion behavior and mechanical toughness of ADH / ALDH@VH in a simulated gastric environment. After incubation at 37°C for 72 hours, the hydrogel... Figure 5 As shown in Figure c, the maximum expansion rate reaches 585%. Figure 8 As shown, its compressive strength gradually decreases during expansion, but the minimum compressive stress still reaches 11 kPa, significantly higher than the physiological pressure in the gastric cavity. Even at maximum expansion, the hydrogel maintains a compressive strength of 11 kPa, far exceeding the physiological pressure within the gastric cavity (2–5 kPa), ensuring its structural integrity in the stomach. Figure 9 As shown. Cyclic testing of the fully expanded hydrogel showed that its performance remained stable after 550 cycles, as indicated. Figure 5 As shown in Figure d, there is no obvious mechanical degradation. Visual observation further confirms this. Figure 5 As shown in Figure e, its morphology did not change significantly before and after loading, and it maintained its original shape even after being continuously compressed under 100g pressure for three days. Figure 10 As shown, it exhibits extremely high mechanical durability.

[0060] Test Example 4

[0061] This test case investigates the adhesive properties of hydrogels.

[0062] Adhesion properties are a key parameter of gastric retention capacity, determined by topological chimerism, interfacial chemistry, and energy dissipation mechanisms. The adhesion properties of ADH / ALDH@VH were evaluated using shear tests. Figure 11 As shown, shear stress is applied by laterally moving two substrates connected by the hydrogel in opposite directions. Hydrogels exhibit strong adhesion to various biological tissues, such as… Figure 5As shown in Figure f, its shear strength in different tissues is: 83.42 kPa in porcine stomach, 47.17 kPa in bovine stomach, 64.79 kPa in porcine skin, and 17.06 kPa in fish skin. To test its biocompatibility, we applied the hydrogel to the skin of a human wrist. After removal, there was no residue, and no irritation, redness, or discomfort was observed, demonstrating its excellent skin compatibility. Notably, this adhesion process requires no surface pretreatment, is completely reversible, and leaves no residue after removal, facilitating its clinical translation.

[0063] Test Example 5

[0064] This test case was used to analyze the biocompatibility of the hydrogel.

[0065] This test case assessed the biocompatibility of the hydrogel with human gastric epithelial cells. The biocompatibility of the ADH / ALDH@VH hydrogel was evaluated using a human gastric mucosal epithelial cell system. Hydrogel powder (10 mg mL⁻¹) was sterilized under UV light and then immersed in complete culture medium at 37°C for 24 h. Cell viability was assessed using the CCK-8 assay (7 × 10³ cells / well, cultured for 24 h), and absorbance was measured at 450 nm. Simultaneously, Calcein-AM / PI live / dead staining was used for supplementary observation of cell morphology and membrane integrity.

[0066] In the apoptosis analysis, GES-1 cells were co-cultured with hydrogel powder for 24 h, followed by Annexin V-FITC / PI double staining. Flow cytometry was used to quantitatively distinguish between viable cells, early apoptotic cells, late apoptotic cells, and necrotic cell populations. All experiments included a blank control with only culture medium and were repeated three times to ensure the statistical reliability of the data.

[0067] Figure 12 Figure a (control group) and Figure 12 Image b (ADH / ALDH@VH group) shows representative fluorescence microscopy images of live / dead staining. Red fluorescence indicates dead cells, green indicates live cells, and the rightmost image is a merged image. In both groups, cells exhibited a healthy morphology with widespread green fluorescence and minimal red signal. These observations suggest that the hydrogel supports cell adhesion and growth without inducing significant cytotoxicity.

[0068] Cell proliferation was further quantitatively analyzed using the CCK-8 assay. The results are as follows: Figure 12 As shown in Figure c, there was no significant difference in cell viability between the control group and the ADH / ALDH@VH treatment group, further confirming that exposure to the hydrogel does not affect cell metabolic activity.

[0069] In addition, we used Annexin V-FITC and propidium iodide (PI) staining combined with flow cytometry to assess apoptosis. Figure 12 The middle d diagram and Figure 12 As shown in Figure e, in the control group, 93.2% of the cells were in a viable state (Q4 quadrant), while the proportion of early and late apoptotic and necrotic cells (Q2+Q3 quadrants) was less than 5.5%. In the hydrogel-treated group, the proportion of viable cells increased slightly (94.4%), while the proportion of apoptotic cells decreased slightly (Q2+Q3 approximately 4.0%), suggesting that the hydrogel does not induce programmed cell death and may even have a certain protective effect.

[0070] In summary, through fluorescence imaging, cell proliferation analysis, and apoptosis detection, we confirmed that the ADH / ALDH@VH hydrogel is non-cytotoxic to gastric epithelial cells and exhibits excellent biocompatibility.

[0071] Test Example 6

[0072] This test case explores the relationship between catalytic performance and hydrogel morphology and structure.

[0073] The catalytic performance of enzyme-loaded hydrogel systems (ADH / ALDH@VH) is influenced by their morphology. Specifically, increasing the specific surface area helps enhance the interaction between the enzyme and the substrate, thereby significantly improving biocatalytic activity. To investigate this effect, we fabricated a series of mesh-structured hydrogels with varying thicknesses to control their specific surface area, such as... Figure 13 As shown in Figure a. Figure 13 As shown in Figure b, the enzyme activity in the 1 mm thick mesh hydrogel is 7 times that of the non-mesh hydrogel, indicating that the effective reaction contact area can improve the efficiency of enzyme immobilization.

[0074] We determined the activities of ADH and ALDH using NADH-coupled UV-Vis spectrophotometry (340 nm). The optimized reaction system had a total volume of 6 mL and contained: 1 M Tris-HCl buffer (pH 8.0), 20 mM NAD⁺, 100 mM acetaldehyde, 3 M KCl solution, 1 M β-mercaptoethanol solution, and ADH and ALDH enzymes. The enzymes were added to a quartz cuvette at 25 °C to initiate the reaction, and absorbance was recorded every 10 s (λ = 340 nm, ε = 6.22 × 10³ M⁻¹ cm⁻¹). Each experiment was repeated three times to ensure data accuracy. This standardized protocol maintains physiological relevance while accurately determining catalytic efficiency.

[0075] The enzyme kinetics of ADH / ALDH@VH were systematically tested. Compared with the free enzyme, the immobilized enzyme exhibited a significantly lower Michaelis constant (Km), indicating a higher affinity for the substrate, while also... Figure 13 As shown in Figure c, the Kcat / Km ratio is significantly improved, reflecting superior catalytic efficiency, as shown in Table 1. To test its time stability, we monitored enzyme activity at 37°C. The activity of the free enzyme decreased rapidly within 12 hours, while the immobilized enzyme maintained relatively high activity under the same conditions, demonstrating the advantages of the hydrogel system. Since the immobilized enzyme is not consumed during catalysis, we also tested its reusability in five consecutive reaction cycles. The immobilized enzyme retained approximately 80% of its initial activity after the fifth cycle, indicating good reproducibility and sustained function.

[0076] Table 1

[0077]

[0078] We further investigated the catalytic activity of the immobilized enzyme under extreme pH conditions, including simulated gastric fluid (pH 1.5–3.5) and alkaline environments (pH 11.5–13.5). Under both extreme conditions, the free enzyme rapidly inactivated, while the immobilized enzyme maintained its activity even after prolonged exposure, indicating that the hydrogel can effectively protect the enzyme from pH-induced denaturation.

[0079] Test Example 7

[0080] This test case investigates the effect of hydrogels on ethanol metabolism.

[0081] To enhance the retention time of immobilized enzymes in the gastric cavity, following Example 1, we further constructed flexible artificial microvilli on the surface of a mesh-structured hydrogel. These microvilli can mechanically interlock with gastric folds, such as... Figure 13 The middle d diagram and Figure 13 As shown in Figure e, this enhances its adhesion to the gastric mucosa. When exposed to simulated gastric juice, the dehydrated hydrogel rapidly absorbs water and restores its original three-dimensional structure. Similar swelling behavior and interlocking phenomena between microvilli and folds were also observed in in vitro experiments on porcine gastric tissue, where the hydrogel rapidly and completely unfolded, effectively achieving gastric retention.

[0082] Finally, we evaluated the effects of ADH / ALDH@VH on ethanol metabolism. In a typical 70 kg adult male, the rate of endogenous ethanol metabolism is approximately 7–10 g / h. Following oral administration of the immobilized enzyme, such as... Figure 13As shown in Figure h, the ethanol clearance rate increased by 30-20% compared to baseline. This significant improvement confirms that the immobilized enzyme accelerates the acetaldehyde conversion process by increasing local enzyme concentration and sustained release. These results establish the in vivo efficacy of this hydrogel-based enzyme delivery platform and demonstrate its therapeutic potential in ethanol metabolism intervention, effectively reducing acetaldehyde exposure and related toxicity.

[0083] In summary, the collagen hydrogel provided by this invention, by combining collagen-based hydrogels with microscale mesh and artificial villous structures, achieves prolonged gastric retention time, enhanced mechanical toughness, and excellent compressive strength under physiological conditions, thus solving the long-standing trade-off between mechanical stability and catalytic efficiency.

[0084] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for preparing collagen hydrogel, characterized in that, The preparation method includes mixing collagen, acrylamide solution, methylenebisacrylamide solution and calcium chloride solution, followed by sonication, then adding oxidant and reducing agent and letting stand.

2. The method for preparing collagen hydrogel according to claim 1, characterized in that, The ratio of collagen, acrylamide solution, methylenebisacrylamide solution and calcium chloride solution is (0.1-0.5) g:(0.8-1.2) mL:(50-150) μL:100 μL.

3. The method for preparing collagen hydrogel according to claim 1 or 2, characterized in that, The acrylamide solution has a mass concentration of 30%-50%; Preferably, the mass concentration of methylenebisacrylamide in the methylenebisacrylamide solution is 10%-30%; Preferably, the molar concentration of calcium chloride in the calcium chloride solution is 2-4 M.

4. The method for preparing collagen hydrogel according to any one of claims 1-3, characterized in that, The oxidant includes any one or a combination of at least two of ammonium persulfate, potassium persulfate, or 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropionylbenzene; Preferably, the molar concentration of the oxidant is 1-3 M; Preferably, the ratio of the oxidant to collagen is (60-80) μL : (0.1-0.5) g; Preferably, the reducing agent comprises any one or a combination of at least two of tetramethylethylenediamine, N,N-dimethyl-p-toluidine, or piperazine; Preferably, the ratio of the reducing agent to collagen is (5-15) μL : (0.1-0.5) g; Preferably, the settling temperature is 0-8℃ and the settling time is 8-20 h.

5. A collagen hydrogel prepared by the method of any one of claims 1-4.

6. The use of the collagen hydrogel according to claim 5 in the preparation of a drug delivery system.

7. An oral delivery system with hangover relief function, characterized in that, The oral delivery system comprises the collagen hydrogel and alcohol-detoxifying enzyme as described in claim 5.

8. The oral delivery system with hangover relief function according to claim 7, characterized in that, The alcohol-detoxifying enzymes include alcohol dehydrogenase and / or acetaldehyde dehydrogenase.

9. The oral delivery system with hangover relief function according to claim 7 or 8, characterized in that, The loading of the alcohol-detoxifying active enzyme is 1%-5% of the collagen hydrogel mass.

10. The use of an oral delivery system with hangover relief function according to any one of claims 7-9 in the preparation of a rapid hangover relief product.

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