Nanocomposite hydrogel adhesive and applications thereof

Abstract

This invention provides a nanocomposite hydrogel adhesive and its applications. The nanocomposite hydrogel adhesive is obtained by mixing and curing a mixture of phase A and phase B. Phase A includes a PDA-MnO2@Gel hybrid nanozyme, photocurable gelatin, a photoinitiator, and an aqueous solvent; phase B includes biodegradable polymer-protected CaO2, a crosslinking agent, and an aqueous solvent. This invention, by introducing the PDA-MnO2@Gel hybrid nanozyme into the hydrogel adhesive, yields a nanocomposite hydrogel adhesive for regulating the inflammatory microenvironment. It exhibits good shape adaptability, tissue adhesion, and biocompatibility, and particularly demonstrates excellent ability to regulate the inflammatory microenvironment. It can effectively remove excess reactive oxygen species from the inflammatory microenvironment, alleviate hypoxia in the affected area, and reduce inflammation levels. This nanocomposite hydrogel adhesive has broad application prospects in the repair of inflammatory wounds.

Description

Technical Field

[0001] This invention belongs to the field of hydrogel preparation technology, specifically relating to a nanocomposite hydrogel adhesive and its application. Background Technology

[0002] In clinical medicine, the rapid healing of surgical and non-surgical wounds in a large number of patients with inflammatory diseases (such as cancer, diabetes, and chronic infected wounds) remains a hot topic and focus of research. The wound sites of patients with inflammatory diseases often possess a unique inflammatory tissue microenvironment, which hinders wound healing. This inflammatory tissue microenvironment is mainly composed of inflammatory cells, inflammatory factors, and biomolecules, and is characterized by low oxygen, low pH, and excessive inflammatory factors. Currently, medical hydrogel adhesives, as a class of medical materials specifically used for adhesion and closure in the medical field, generally have the advantages of being easy to use and safe and reliable, effectively meeting the needs of surgical and non-surgical wound closure. However, current hydrogel adhesives typically only function as a sealant and lack the ability to regulate the specific inflammatory microenvironment of the affected area, thus limiting their healing-promoting function. Effectively regulating the inflammatory microenvironment of the affected area is crucial for the healing and recovery of inflammatory wounds and for inhibiting scar formation.

[0003] Currently, the healing and repair of inflammatory wounds generally involves separate treatments of anti-inflammatory drugs and wound closure. However, anti-inflammatory treatment using antibiotics or hormones makes wound care procedures cumbersome, prolongs the treatment period, and is prone to various side effects. Furthermore, the medical hydrogel adhesives used for wound closure have limited functionality, cannot effectively regulate the inflammatory microenvironment at the wound site, and still have limitations in terms of safety.

[0004] Reactive oxygen species (ROS), as inflammatory mediators, are overexpressed in the inflammatory microenvironment. Furthermore, excessive ROS can further induce oxidative stress damage in cells, exacerbating the difficulty of healing inflammatory wounds. Therefore, to promote rapid healing of inflammatory wounds, it is necessary to develop materials that can consume ROS, reduce oxidative stress, and remodel the inflammatory microenvironment. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a nanocomposite hydrogel adhesive and its application. The nanocomposite hydrogel adhesive not only seals inflammatory wounds but also consumes reactive oxygen species, reduces oxidative stress, and remodels the inflammatory microenvironment, thereby accelerating scarless healing of inflammatory wounds.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a nanocomposite hydrogel adhesive, which is obtained by mixing and curing phase A and phase B;

[0008] Phase A comprises PDA-MnO2@Gel hybrid nanozyme, photocurable gelatin, photoinitiator, and aqueous solvent;

[0009] Phase B comprises biodegradable polymer-protected CaO2, a crosslinking agent, and an aqueous solvent;

[0010] The nanocomposite hydrogel adhesive has a structure with a dual cross-linked network.

[0011] Preferably, the A phase comprises, by mass fraction, 0.001–0.05 wt% PDA-MnO2@Gel hybrid nanozyme, 15–30 wt% photocurable gelatin, 0.5–1.5 wt% photoinitiator, and the balance being an aqueous solvent.

[0012] Preferably, the PDA-MnO2@Gel hybrid nanozyme is prepared by a one-step synthesis method using gelatin, dopamine hydrochloride, and potassium permanganate.

[0013] Preferably, the mass ratio or molar ratio of the gelatin, dopamine hydrochloride and potassium permanganate is 2:(0.8-1.5):(0.2-0.5).

[0014] Preferably, the particle size of the PDA-MnO2@Gel hybrid nanozyme is 30-200 nm.

[0015] Preferably, the photocurable gelatin is selected from any one or more of glycidyl methacrylate modified gelatin, methacrylated gelatin, or methacrylated gelatin.

[0016] Preferably, the photoinitiator is selected from one or more of phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone (I2959), or camphorquinone.

[0017] Preferably, the aqueous solvent in phase A is selected from one or more of water, physiological saline, or buffer solutions.

[0018] Preferably, the B phase comprises, by mass fraction, 0.001–0.01 wt% CaO2 protected by a biodegradable polymer, 10–30 wt% oxidized dextran, and the balance being an aqueous solvent.

[0019] Preferably, the degradable polymer in the CaO2 protected by the degradable polymer is selected from any one or more of polycaprolactone, polylactic acid, or polylactic acid-polyglycolic acid copolymer;

[0020] Preferably, the particle size of the CaO2 in the biodegradable polymer-protected CaO2 is 20–100 nm.

[0021] Preferably, the crosslinking agent is selected from one or more of oxidized dextran, oxidized glucomannan, or oxidized guar gum.

[0022] Preferably, the molecular weight of the dextran in the oxidized dextran is 30-50 kDa.

[0023] Preferably, the aqueous solvent in phase B is selected from one or more of water, physiological saline, or buffer solutions.

[0024] Preferably, the volume ratio of phase A to phase B is 1:(0.8 to 1.5).

[0025] Secondly, the present invention provides an application of the above-mentioned nanocomposite hydrogel adhesive in the field of inflammatory wound treatment.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0027] This invention provides a nanocomposite hydrogel adhesive, which is obtained by mixing and curing phase A and phase B. In this invention, phase A includes PDA-MnO2@Gel hybrid nanozyme, photocurable gelatin, photoinitiator, and aqueous solvent; phase B includes biodegradable polymer-protected CaO2, crosslinking agent, and aqueous solvent.

[0028] The PDA-MnO2@Gel hybrid nanozyme can be efficiently and rapidly prepared in pure aqueous solution using a one-step synthesis method. The final product is a hybrid spherical structure composed of gelatin, polydopamine, and dispersed manganese dioxide nanodots, integrating the advantages of oxidative polymerization and biomineralization. Gelatin acts as a biomineralization template. The hybrid spherical structure of the PDA-MnO2@Gel hybrid nanozyme endows it with multiple reactive oxygen species scavenging capabilities exhibiting cascade catalytic effects, including highly efficient superoxide dismutase (SOD)-like activity, catalase-like activity, and the ability to scavenge free radicals. It can completely convert various forms of reactive oxygen species, such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals, into harmless water and oxygen.

[0029] In this invention, photocurable gelatin and crosslinking agent form a nanocomposite hydrogel adhesive matrix through a double crosslinking network. The crosslinking agent and photocurable gelatin first form a first layer of dynamic covalent crosslinking network, which can maintain the injectability of the nanocomposite hydrogel adhesive. After being applied to the affected area, the double bonds on the photocurable gelatin can be polymerized by a photoinitiator under ultraviolet irradiation to form a second layer of covalent crosslinking network, enabling the nanocomposite hydrogel adhesive to be rapidly formed and firmly adhered.

[0030] In this invention, the PDA-MnO2@Gel hybrid nanoenzyme, as the main component exerting antioxidant and anti-inflammatory effects, is incorporated into the hydrogel matrix within the nanocomposite hydrogel adhesive system. Its free amino groups can also form dynamic covalent bonds with the active groups on the crosslinking agent, enhancing the mechanical properties of the nanocomposite hydrogel adhesive to a certain extent. Simultaneously, the PDA-MnO2@Gel hybrid nanoenzyme can also assist CaO2 in fully releasing oxygen. Furthermore, the alkaline calcium hydroxide produced by the slow decomposition of CaO2 in water can also help to transform the low pH of the inflammatory microenvironment into a normal physiological pH. Therefore, the nanocomposite hydrogel adhesive provided by this invention possesses good shape adaptability, tissue adhesion, and biocompatibility, and in particular, exhibits excellent ability to regulate the inflammatory microenvironment. It can effectively remove excess reactive oxygen species from the inflammatory microenvironment, alleviate hypoxia in the affected area, and reduce inflammation levels, showing broad application prospects in the repair of inflammatory wounds. Attached Figure Description

[0031] Figure 1 A diagram illustrating the formation mechanism of the nanocomposite hydrogel adhesive provided by this invention;

[0032] Figure 2 TEM image and elemental distribution map of the PDA-MnO2@Gel hybrid nanozyme obtained in Example 1;

[0033] Figure 3 The graph shows the SOD-like enzyme activity results of the PDA-MnO2@Gel hybrid nanozyme obtained in Example 1 at different concentrations.

[0034] Figure 4 The graph shows the activity results of the PDA-MnO2@Gel hybrid nanozyme obtained in Example 1 at different concentrations, similar to the CAT enzyme.

[0035] Figure 5 The figures show the cytotoxicity test results of the nanocomposite hydrogel adhesives obtained in Examples 1-4 and the hydrogel adhesive in Comparative Example 1. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] To address the problem that existing medical hydrogel adhesives used for wound closure have limited functionality and cannot effectively regulate the inflammatory microenvironment at the site of inflammatory wounds, this invention provides a nanocomposite hydrogel adhesive, which is obtained by mixing and curing phase A and phase B.

[0038] In this invention, phase A comprises PDA-MnO2@Gel hybrid nanozyme, photocurable gelatin, photoinitiator, and aqueous solvent. In some embodiments of this invention, phase A comprises, by mass fraction, 0.001–0.05 wt% PDA-MnO2@Gel hybrid nanozyme, 15–30 wt% photocurable gelatin, 0.5–1.5 wt% photoinitiator, with the balance being aqueous solvent; preferably, it comprises 0.005–0.03 wt% PDA-MnO2@Gel hybrid nanozyme, 20–25 wt% photocurable gelatin, 0.5–1 wt% photoinitiator, with the balance being aqueous solvent.

[0039] In this invention, the PDA-MnO2@Gel hybrid nanozyme is prepared by a one-step synthesis method using gelatin, dopamine hydrochloride, and potassium permanganate. It possesses multiple reactive oxygen species scavenging capabilities with cascade catalytic effects, exhibiting antioxidant and anti-inflammatory properties. It can polymerize with the crosslinking agent in phase B, thereby enhancing the mechanical properties of the nanocomposite hydrogel adhesive to a certain extent. The preferred mass ratio of gelatin, dopamine hydrochloride, and potassium permanganate is 2:(0.8–1.5):(0.2–0.5), more preferably 2:(1–1.2):(0.3–0.5), and most preferably 2:1:0.3. The gelatin is preferably type A gelatin derived from pigskin. In some embodiments of this invention, the gelatin is preferably dissolved in water, preferably ultrapure water, and stirred at 40–60°C for 15–40 min, preferably at 50°C for 20 min. Subsequently, an aqueous solution of dopamine hydrochloride is added, and the mixture is stirred for 15–40 min under sealed conditions at 40–60°C, preferably 50°C. Finally, an aqueous solution of potassium permanganate is added, and the reaction is stirred for another 30–50 min to obtain the PDA-MnO2@Gel hybrid nanozyme. Preferably, after the reaction is complete, the obtained product is dialyzed using an 8–14 kDa dialysis bag for 2–3 days and then lyophilized for later use. The particle size of the PDA-MnO2@Gel hybrid nanozyme prepared by this invention is preferably 30–200 nm, more preferably 50–120 nm.

[0040] The gelatin in the PDA-MnO2@Gel hybrid nanozyme acts as a biomineralization template. Testing revealed that the PDA-MnO2@Gel hybrid nanozyme exhibits highly efficient superoxide dismutase-like activity, catalase-like activity, and free radical scavenging capabilities. It can completely convert various forms of reactive oxygen species, such as superoxide radicals, hydrogen peroxide, and hydroxyl radicals, into harmless water and oxygen, demonstrating multiple reactive oxygen species scavenging abilities. This highly efficient reactive oxygen species scavenging ability with cascade catalysis is attributed to the hybrid structure of polydopamine and manganese dioxide in the PDA-MnO2@Gel hybrid nanozyme.

[0041] In this invention, the photocurable gelatin is obtained by photocuring and molecularly modifying gelatin. It can form a nanocomposite hydrogel adhesive matrix with the crosslinking agent in phase B through a double crosslinking network. Specifically, it is preferably selected from any one or more of glycidyl methacrylate-modified gelatin, methacrylated gelatin, or methacrylated gelatin, with glycidyl methacrylate-modified gelatin (abbreviated as "Gel-GMA") being the most preferred. This invention does not impose any particular limitation on the preparation method of the photocurable gelatin; it can be carried out using methods well known to those skilled in the art. In some embodiments of this invention, the photocurable gelatin is preferably prepared according to the following method:

[0042] Gelatin is dissolved in a buffer solution, preferably DPBS buffer, with a pH of 5-6, and stirred at 40-60°C, preferably 50°C, until completely dissolved. The photocurable molecule is then added to the solution and stirred at 40-60°C, preferably 50°C, for 3-5 hours. The resulting product is precipitated in ethanol, preferably 4-5 times its volume of ethanol. The precipitated product is dissolved in a small amount of water, purified by dialyzing for 2-3 days, and then lyophilized to obtain the photocurable gelatin, which is then stored sealed at room temperature for later use.

[0043] In this invention, the photoinitiator is used to initiate the self-polymerization of photocurable gelatin under ultraviolet conditions. Specifically, it can be selected from any one or more of phenyl-2,4,6-trimethylbenzoyl phosphite (LAP), 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone (I2959), or camphorquinone, preferably phenyl-2,4,6-trimethylbenzoyl phosphite (LAP). This invention does not have particular limitations on the source of the photoinitiator; commercially available products are sufficient.

[0044] In this invention, the aqueous solvent is selected from any one or more of water, physiological saline or buffer solution, preferably water, which can be any one or more of deionized water, ultrapure water or distilled water.

[0045] In this invention, the B phase comprises biodegradable polymer-protected CaO2, a crosslinking agent, and an aqueous solvent. In some embodiments of this invention, the B phase comprises, by mass fraction, 0.001–0.01 wt% (preferably 0.005–0.008 wt%) of biodegradable polymer-protected CaO2, 10–30 wt% (preferably 15–20 wt%) of oxidized dextran, with the balance being an aqueous solvent.

[0046] In this invention, the CaO2 protected by the biodegradable polymer is hydrophobically protected by CaO2 loaded into microspheres of the biodegradable polymer to prevent CaO2 from decomposing upon contact with water during preparation and generating oxygen beforehand. In this invention, the biodegradable polymer is preferably one or more of polycaprolactone (PCL), polylactic acid (PLA), or polylactic acid-polyglycolic acid copolymer (PLGA), with polycaprolactone being the most preferred. In this invention, the particle size of CaO2 in the biodegradable polymer-protected CaO2 is 20–100 nm, preferably 50–80 nm. In some embodiments of this invention, taking polycaprolactone as an example, polycaprolactone-protected CaO2 can be prepared by the following method:

[0047] Weigh CaO2 and ultrasonically disperse it in chloroform for 10–20 minutes. Then, add polycaprolactone to dissolve it, obtaining a CaO2 chloroform dispersion. Weigh polyvinyl alcohol (type 1788) and dissolve it in deionized water. Stir and dissolve it at 50–60°C, then cool to room temperature to obtain a polyvinyl alcohol aqueous solution. Add the CaO2 chloroform dispersion dropwise to the CaO2 chloroform dispersion under vigorous stirring, and continue stirring at 40–50°C for 3–4 hours. Then, obtain polycaprolactone-protected CaO2 by filtration and drying.

[0048] When the degradable polymer is PLA or PLGA, PLA or PLGA-protected CaO2 can be prepared using the method described above. This invention does not impose any particular limitations on the preparation method of CaO2; any method well-known to those skilled in the art can be used.

[0049] In this invention, the biodegradable polymer-protected CaO2, after being cured under ultraviolet light at the affected area (i.e., the inflammatory wound environment), gradually decomposes to produce oxygen through the following process. The PDA-MnO2@Gel hybrid nanozyme can assist CaO2 in fully releasing oxygen. In addition, the alkaline calcium hydroxide produced by the slow decomposition of CaO2 in water can also change the low pH of the inflammatory wound environment to a normal physiological pH.

[0050] Cao2+2H2O→Ca(OH)2+H2O2

[0051]

[0052] In this invention, the crosslinking agent plays a crosslinking role and is specifically selected from any one or more of oxidized dextran, oxidized glucomannan, or oxidized guar gum, preferably oxidized dextran. In this invention, the oxidized dextran is obtained by treating dextran with an oxidizing agent, and the molecular weight of the dextran is preferably 30-50 kDa, more preferably 40 kDa. The oxidizing agent is selected from any one or more of sodium periodate, potassium periodate, or periodic acid. In some embodiments of this invention, oxidized dextran is preferably prepared according to the following method:

[0053] Dextran was dissolved in ultrapure water and stirred for 10–20 minutes. Then, an oxidant was added, and the reaction was continued at room temperature in the dark for one day. After the reaction was completed, ethylene glycol was added to neutralize the remaining oxidant, and the mixture was stirred for another 2–4 hours. The solution was then dialyzed through a 3 kDa dialysis bag for 2 days and lyophilized to obtain a white powdery oxidized dextran.

[0054] In this invention, the aqueous solvent in phase B is selected from any one or more of water, physiological saline or buffer solution, preferably water, which can be any one or more of deionized water, ultrapure water or distilled water.

[0055] This invention introduces a PDA-MnO2@Gel hybrid nanozyme into a hydrogel binder, resulting in a nanocomposite hydrogel adhesive for regulating the inflammatory microenvironment. The nanocomposite hydrogel exhibits good shape adaptability, tissue adhesion, and biocompatibility, and particularly demonstrates excellent ability to regulate the inflammatory microenvironment. It can effectively remove excess reactive oxygen species from the inflammatory microenvironment, alleviate hypoxia in the affected area, and reduce inflammation levels. Therefore, the nanocomposite hydrogel adhesive of this invention has broad application prospects in the repair of inflammatory wounds.

[0056] This invention also provides a method for preparing the above-mentioned nanocomposite hydrogel adhesive, the formation mechanism of which is shown in the schematic diagram below. Figure 1 As shown, the specific steps include:

[0057] S1: Provides phase A and phase B;

[0058] S2: After mixing phase A and phase B evenly, the mixture is cured under ultraviolet light to obtain a nanocomposite hydrogel adhesive.

[0059] According to the present invention, phase A and phase B are first provided. In some embodiments of the present invention, phase A is obtained by dissolving PDA-MnO2@Gel hybrid nanozyme, photocurable gelatin, and a photoinitiator in an aqueous solvent. The proportions of PDA-MnO2@Gel hybrid nanozyme, photocurable gelatin, photoinitiator, and aqueous solvent are as described in the relevant content of the above technical solutions and will not be repeated here. Phase B is obtained by dissolving biodegradable polymer-protected CaO2 and a crosslinking agent in an aqueous solvent. The proportions of biodegradable polymer-protected CaO2, crosslinking agent, and aqueous solvent are as described in the relevant content of the above technical solutions and will not be repeated here.

[0060] Then, phase A and phase B are mixed at a volume ratio of 1:(0.8-1.5), preferably 1:1. After uniform mixing, the mixture is cured under ultraviolet light to obtain the nanocomposite hydrogel adhesive. In some embodiments of the present invention, the ultraviolet wavelength for curing is 365 nm, and the curing time is 1-5 min.

[0061] Testing revealed that the nanocomposite hydrogel adhesive reduced the expression of the pro-inflammatory factor TNF-α and increased the expression of the anti-inflammatory factor IL-10 in LPS-stimulated mouse fibroblasts, indicating that the nanocomposite hydrogel adhesive possesses excellent anti-inflammatory effects. Furthermore, the nanocomposite hydrogel adhesive exhibits no cytotoxicity and excellent biocompatibility. Based on this, the present invention also provides an application of the above-mentioned nanocomposite hydrogel adhesive in the field of inflammatory wound treatment.

[0062] To further illustrate the present invention, detailed descriptions are provided below through the following embodiments. All experimental materials used in the following embodiments of the present invention are commercially available products. Specifically, the gelatin was purchased from SIGMA-ALDRICH, model number V900863; the mouse fibroblasts L929 were provided by the ATCC Preservation Center.

[0063] Preparation Example 1

[0064] This preparation example provides a PDA-MnO2@Gel hybrid nanozyme, hydrophobically protected CaO2, photocurable gelatin (Gel-GMA), and oxidized dextran, and the preparation method is as follows:

[0065] (1) Preparation of PDA-MnO2@Gel hybrid nanozyme: 200 mg of gelatin was dissolved in 100 mL of ultrapure water and stirred at 50 °C for 20 min. Then, 1 mL of an aqueous solution containing 100 mg of dopamine hydrochloride was added at room temperature with stirring, and the reaction was continued with stirring for 15 min. Next, 1 mL of an aqueous solution containing 30 mg of potassium permanganate was added, and the reaction was continued with stirring for 30 min. After that, the reaction was stopped, and the product was dialyzed through an 8-14 kDa dialysis bag for 3 days and then lyophilized for later use.

[0066] (2) Preparation of hydrophobically protected CaO2: 3g of calcium chloride was dissolved in 30mL of ultrapure water, followed by the addition of 15mL of 1M ammonia solution and stirring for 5min. Then, 120mL of PEG200 was added and stirring continued for 30min. Finally, 15mL of 30% hydrogen peroxide was added dropwise to the solution, and stirring continued for 2 hours to obtain a milky white solution. Next, 100mL of 1M sodium hydroxide was added to adjust the pH to 11-12. The solution was then centrifuged, washed, and dried to obtain a slightly yellow powdered CaO2. To slow down the hydrolysis rate of CaO2 upon contact with water, it can be protected by loading CaO2 into biodegradable polymer microspheres.

[0067] (3) Preparation of photocurable gelatin (Gel-GMA) under acidic conditions: 5 g of gelatin was added to 50 mL of DPBS buffer (pH adjusted to 5 with 1 M HCl) and stirred at 50 °C until completely dissolved; then 5 mL of GMA was added to the solution, and the reaction mixture was stirred at 50 °C for 3 hours. The product was precipitated in ethanol (five times the volume in excess) at room temperature and kept overnight with gentle stirring to further extract unreacted GMA. The functionalized gelatin (Gel-GMA) was dissolved in a small amount of water, purified by dialyzing for 2 days, and lyophilized for later use.

[0068] (4) Preparation of oxidized dextran by strong oxidation modification: 10g of dextran was added to 100mL of ultrapure water and stirred to dissolve for 10min. Then, 2g of sodium periodate was added and the reaction was continued at room temperature in the dark for one day. After the reaction was completed, 10mL of ethylene glycol was added to neutralize the remaining oxidant, and stirring was continued for 2 hours. The mixed solution was then dialyzed through a 3kDa dialysis bag for 2 days and freeze-dried to obtain white powdered oxidized dextran.

[0069] The prepared PDA-MnO2@Gel hybrid nanozyme, hydrophobically protected CaO2, photocurable gelatin (Gel-GMA), and oxidized dextran were used in the following examples and comparative examples.

[0070] The surface morphology of the PDA-MnO2@Gel hybrid nanozyme obtained in Example 1 was characterized using scanning electron microscopy. The obtained TEM images and elemental distribution maps are shown below. Figure 2 As shown, the PDA-MnO2@Gel hybrid nanozyme exhibits a hybrid spherical structure, with ultra-small manganese dioxide nanodots mixed within spherical nanoparticles composed of gelatin and polydopamine.

[0071] The SOD-like enzyme activity and CAT-like enzyme activity of the PDA-MnO2@Gel hybrid nanozyme obtained in Preparation Example 1 were evaluated using the following methods:

[0072] Evaluation of SOD-like enzyme activity:

[0073] The SOD-like activity of PDA-MnO2@Gel hybrid nanozymes was evaluated by measuring their inhibitory effect on NBT photoreduction. PBS (0.01M, pH 7.3), methionine solution (130mM), riboflavin solution (0.2mM), NBT solution (0.75mM), and PDA@MnO2@Gel hybrid nanozyme solutions at different concentrations (0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, and 0.5mg / mL) were mixed at a volume ratio of 6:1:1:1:1 and irradiated at room temperature for 10 minutes under a constant light intensity lamp to obtain five experimental groups. The absorbance of the five experimental groups at 560nm was then measured. A mixture of PBS (0.01M, pH 7.3), methionine solution (130mM), riboflavin solution (0.2mM), NBT solution (0.75mM), and PDA@MnO2@Gel hybrid nanozyme solutions at different concentrations (0.05mg / mL, 0.1mg / mL, 0.2mg / mL, 0.3mg / mL, 0.4mg / mL, 0.5mg / mL) was prepared at a volume ratio of 6:1:1:1:1. Samples without light exposure served as negative controls. A mixture of PBS (0.01M, pH 7.3), methionine solution (130mM), riboflavin solution (0.2mM), and NBT solution (0.75mM) at a volume ratio of 7:1:1:1 (containing no PDA@MnO2@Gel hybrid nanozyme) was prepared and then exposed to light, serving as a positive control sample. ·- The clearance rate is calculated using the following formula:

[0074] I = (1 - (A0 - A) n ) / (A p -A n ))×100%;

[0075] Where I is O2 ·- The clearance rate, A0 is the absorbance of the sample, A n The absorbance of the negative control, A p The absorbance is for the positive control.

[0076] Test results are as follows Figure 3 As shown, the PDA-MnO2@Gel hybrid nanozyme can catalyze the removal of O2 from the reaction system. ·- Furthermore, as the material concentration increases, its O2... ·- The scavenging efficiency is significantly improved. Therefore, the PDA-MnO2@Gel hybrid nanozyme exhibits highly efficient SOD-like enzyme activity.

[0077] Evaluation of CAT-like enzyme activity:

[0078] The CAT-like enzyme activity of PDA-MnO2@Gel hybrid nanozymes was evaluated by measuring their ability to catalyze the production of oxygen from hydrogen peroxide. The oxygen content in the solution was detected using an oxygen probe ([Ru(dpp)3]Cl2), as its fluorescence can be dynamically quenched by oxygen molecules. PBS (0.01M, pH 7.3), [Ru(dpp)3]Cl2 solution (10 μM), H2O2 solution (100 mM), and different concentrations of PDA@MnO2@Gel hybrid nanozyme solutions were mixed in a 7:1:1:1 volume ratio and incubated at 37°C in the dark for 10 minutes to obtain the experimental group samples. The control group samples were prepared by using ultrapure water instead of the PDA@MnO2@Gel hybrid nanozyme solution. The fluorescence spectra of the experimental and control group samples at 600–650 nm and the fluorescence intensity at 610 nm were recorded using a fluorescence spectrophotometer under excitation at 455 nm. The oxygen generation efficiency was calculated using the following formula:

[0079] Oxygen generation efficiency = (1-I s / I0)×100%;

[0080] Where I0 is the fluorescence intensity of the control group sample, I s The fluorescence intensity of the experimental group samples is shown.

[0081] Test results are as follows Figure 4 As shown, the fluorescence intensity of the reaction system significantly decreased with the addition of PDA-MnO2@Gel hybrid nanozymes, indicating that PDA-MnO2@Gel hybrid nanozymes can catalyze the rapid decomposition of hydrogen peroxide to produce oxygen, and a strong catalytic effect can be achieved even at a low concentration. Therefore, PDA-MnO2@Gel hybrid nanozymes possess highly efficient CAT-like enzyme activity.

[0082] Example 1

[0083] This embodiment provides a nanocomposite hydrogel adhesive, the formation mechanism of which is shown in the figure below. Figure 1 As shown; the specific preparation method is as follows:

[0084] (1) PDA-MnO2@Gel hybrid nanozyme and Gel-GMA were dissolved in a solution containing 1 wt% photoinitiator LAP to form phase A. The concentration of PDA-MnO2@Gel hybrid nanozyme was 0.005 wt%, and the concentration of Gel-GMA was 25 wt%.

[0085] (2) Hydrophobically protected CaO2 and oxidized dextran were dissolved in secondary water to form phase B. The concentration of hydrophobically protected CaO2 was 0.005 wt%, and the concentration of oxidized dextran was 20 wt%.

[0086] (3) Mix phase A and phase B in a 1:1 ratio and cure by UV irradiation for 1 minute to obtain the nanocomposite hydrogel adhesive of the present invention.

[0087] Example 2

[0088] This embodiment provides a nanocomposite hydrogel adhesive, the formation mechanism of which is shown in the figure below. Figure 1 As shown;

[0089] The specific preparation method is as follows:

[0090] (1) PDA-MnO2@Gel hybrid nanozyme and Gel-GMA were dissolved in a solution containing 1 wt% photoinitiator LAP to form phase A. The concentration of PDA-MnO2@Gel hybrid nanozyme was 0.01 wt%, and the concentration of Gel-GMA was 25 wt%.

[0091] (2) Hydrophobically protected CaO2 and oxidized dextran were dissolved in secondary water to form phase B. The concentration of hydrophobically protected CaO2 was 0.005 wt%, and the concentration of oxidized dextran was 20 wt%.

[0092] (3) Mix phase A and phase B in a 1:1 ratio and cure by UV irradiation for 1 minute to obtain the nanocomposite hydrogel adhesive of the present invention.

[0093] Example 3

[0094] This embodiment provides a nanocomposite hydrogel adhesive, the formation mechanism of which is shown in the figure below. Figure 1 As shown;

[0095] The specific preparation method is as follows:

[0096] (1) PDA-MnO2@Gel hybrid nanozyme and Gel-GMA were dissolved in a solution containing 1 wt% photoinitiator LAP to form phase A. The concentration of PDA-MnO2@Gel hybrid nanozyme was 0.03 wt%, and the concentration of Gel-GMA was 25 wt%.

[0097] (2) Hydrophobically protected CaO2 and oxidized dextran were dissolved in secondary water to form phase B. The concentration of hydrophobically protected CaO2 was 0.005 wt%, and the concentration of oxidized dextran was 20 wt%.

[0098] (3) Mix phase A and phase B in a 1:1 ratio and cure by UV irradiation for 1 minute to obtain the nanocomposite hydrogel adhesive of the present invention.

[0099] Example 4

[0100] This embodiment provides a nanocomposite hydrogel adhesive, the formation mechanism of which is shown in the figure below. Figure 1 As shown; the specific preparation method is as follows:

[0101] (1) PDA-MnO2@Gel hybrid nanozyme and Gel-GMA were dissolved in a solution containing 1 wt% photoinitiator LAP to form phase A. The concentration of PDA-MnO2@Gel hybrid nanozyme was 0.05 wt%, and the concentration of Gel-GMA was 25 wt%.

[0102] (2) Hydrophobically protected CaO2 and oxidized dextran were dissolved in secondary water to form phase B. The concentration of hydrophobically protected CaO2 was 0.005 wt%, and the concentration of oxidized dextran was 20 wt%.

[0103] (3) Mix phase A and phase B in a 1:1 ratio and cure by UV irradiation for 1 minute to obtain the nanocomposite hydrogel adhesive of the present invention.

[0104] Comparative Example 1

[0105] This comparative example provides a hydrogel adhesive, the preparation method of which is as follows:

[0106] (1) Gel-GMA was dissolved in a solution containing 1 wt% photoinitiator LAP to form phase A. The concentration of Gel-GMA was 25 wt%.

[0107] (2) Hydrophobically protected CaO2 and oxidized dextran were dissolved in secondary water to form phase B. The concentration of hydrophobically protected CaO2 was 0.005 wt%, and the concentration of oxidized dextran was 20 wt%.

[0108] (3) Mix phase A and phase B in a 1:1 ratio, and cure by UV light for 1 minute to form control sample 1.

[0109] Comparative Example 2

[0110] This comparative example provides a hydrogel adhesive, the preparation method of which is as follows:

[0111] (1) PDA-MnO2@Gel hybrid nanozyme and Gel-GMA were dissolved in a solution containing 1 wt% photoinitiator LAP to form phase A. The concentration of PDA-MnO2@Gel hybrid nanozyme was 0.01 wt%, and the concentration of Gel-GMA was 25 wt%.

[0112] (2) Oxidized dextran was dissolved in deionized water to form phase B. The concentration of oxidized dextran was 20 wt%.

[0113] (3) Mix phase A and phase B in a 1:1 ratio, and cure under ultraviolet light for 1 minute to form control sample 2.

[0114] Cytotoxicity test

[0115] The cytotoxicity of the products from Examples 1-4 was tested using the following methods:

[0116] Mouse fibroblast L929 cells were seeded into 96-well plates and cultured adherently for 24 hours at a cell density of 1×10⁶ cells / well. 5 Cells / mL. Control group cells were cultured in fresh culture medium; experimental group cells were cultured in extracts of the products from Examples 1-4 for 24 hours. Each group had 6 replicates. After treatment, each well of cells was washed three times with PBS, and then treated with extracts containing Calcein-AM (2 μmol / L). -1 ) and propidium iodide (PI, 4 μmol L) -1 The cells were stained with a mixture of 1,2 ...

[0117] The extracts of the products in Examples 1-4 were obtained by extracting 1 mL of the corresponding product from 5 mL of complete culture medium for 36 hours.

[0118] Test results are as follows Figure 5 As shown, the nanocomposite hydrogel of the present invention hardly causes cell death and has good cell compatibility.

[0119] Anti-inflammatory effect test

[0120] The anti-inflammatory effects of the products from Examples 1-4 and Comparative Examples 1-2 were tested using the following methods:

[0121] Mouse fibroblast L929 cells were seeded into 96-well plates and cultured adherently for 24 hours at a cell density of 1×10⁶ cells / well. 5 Cells were cultured at a density of 1 cell / mL. Control group cells were cultured in fresh medium; LPS group cells were cultured in fresh medium containing 20 μg / mL LPS for 2 hours; experimental group cells were first cultured in fresh medium containing 20 μg / mL LPS for 2 hours, then the medium was replaced with extracts from different Examples 1-4 and Comparative Examples 1-2 and cultured for 24 hours. Each group had six replicates. The cell culture supernatant was then collected from each group, and quantifications of TNF-α and IL-10 levels were obtained using an ELISA kit for TNF-α and IL-10, following the kit's instructions.

[0122] The extracts of the products of Examples 1-4 and Comparative Examples 1-2 were obtained by extracting 1 mL of the corresponding product from 5 mL of complete culture medium for 36 hours.

[0123] The test results are shown in Table 1 below:

[0124] Table 1

[0125]

[0126] As shown in Table 1, the extracts of the products from Examples 1-4 all reduced the expression of the pro-inflammatory factor TNF-α and increased the expression of the anti-inflammatory factor IL-10 in LPS-stimulated cells. However, the anti-inflammatory effect of the extract from Comparative Example 1, which lacked the PDA-MnO2@Gel hybrid nanozyme, was not significant. Compared to Example 2, Comparative Example 2 lacked calcium peroxide in phase B, resulting in a slightly lower anti-inflammatory efficiency. This indicates that the nanocomposite hydrogel adhesive provided by this invention can effectively alleviate LPS-induced cellular inflammatory responses.

[0127] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A nanocomposite hydrogel adhesive, characterized by, It is obtained by mixing and curing phase A and phase B; Phase A comprises PDA-MnO2@Gel hybrid nanozyme, photocurable gelatin, photoinitiator, and aqueous solvent; Phase B comprises biodegradable polymer-protected CaO2, a crosslinking agent, and an aqueous solvent; The nanocomposite hydrogel adhesive has a structure with a dual cross-linked network.

2. The nanocomposite hydrogel adhesive of claim 1, wherein, The A phase comprises, by mass fraction, 0.001-0.05 wt% PDA-MnO2@Gel hybrid nanozyme, 15-30 wt% photocurable gelatin, 0.5-1.5 wt% photoinitiator, and the balance being an aqueous solvent.

3. The nanocomposite hydrogel adhesive of claim 1 or 2, wherein, The PDA-MnO2@Gel hybrid nanozyme was prepared by a one-step synthesis method using gelatin, dopamine hydrochloride, and potassium permanganate. The mass ratio or molar ratio of the gelatin, dopamine hydrochloride, and potassium permanganate is 2:(0.8~1.5):(0.2~0.5).

4. The nanocomposite hydrogel adhesive of claim 3, wherein, The particle size of the PDA-MnO2@Gel hybrid nanozyme is 30~200 nm.

5. The nanocomposite hydrogel adhesive of claim 1 or 2, wherein, The photocurable gelatin is selected from any one or more of glycidyl methacrylate modified gelatin, methacrylated gelatin, or methacrylated gelatin. The photoinitiator is selected from any one or more of phenyl-2,4,6-trimethylbenzoyl lithium phosphite, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylphenylacetone, or camphorquinone; The aqueous solvent in phase A is selected from any one or more of water, physiological saline, or buffer solutions.

6. The nanocomposite hydrogel adhesive of claim 1 or 2, wherein, The B phase, by mass fraction, comprises 0.001-0.01 wt% CaO2 protected by a biodegradable polymer, 10-30 wt% oxidized dextran, and the balance being an aqueous solvent.

7. The nanocomposite hydrogel adhesive of claim 6, wherein, The degradable polymer in the CaO2 protected by the degradable polymer is selected from any one or more of polycaprolactone, polylactic acid, or polylactic acid-polyglycolic acid copolymer. The CaO2 protected by the biodegradable polymer has a particle size of 20~100 nm.

8. The nanocomposite hydrogel adhesive of claim 6, wherein, The crosslinking agent is selected from any one or more of oxidized dextran, oxidized glucomannan, or oxidized guar gum; The molecular weight of the dextran in the oxidized dextran is 30~50 kDa; The aqueous solvent in phase B is selected from any one or more of water, physiological saline, or buffer solutions.

9. The nanocomposite hydrogel adhesive according to claim 6, characterized in that, The volume ratio of phase A to phase B is 1:(0.8~1.5).

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