Nanoprotease functionalized dynamic hydrogel and preparation method and application thereof

Abstract

The present application relates to the field of biological medicine and tissue engineering, and particularly relates to a nano-enzyme functionalized dynamic hydrogel as well as a preparation method and application thereof.The nano-enzyme functionalized dynamic hydrogel comprises black phosphorus nanosheets and nano-enzymes.The preparation method of the nano-enzyme functionalized dynamic hydrogel comprises the following steps: (1) synthesis of a hydrogel precursor solution; (2) preparation of biomimetic nano-enzymes; and (3) preparation of a dynamic hydrogel.In the present application, BP can maintain Ce(III) / Ce(IV) redox cycling through self-oxidation and improve the ROS scavenging capacity in the microenvironment, thereby serving as CeO x a stabilizer of nano-enzymes.In addition, the MBC obtained by wrapping BP@CeO x in the nucleus pulposus cell membrane can better achieve nucleus pulposus cell targeting, so that BP@CeO x can effectively and durably scavenge intracellular ROS.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine and tissue engineering, and particularly relates to a nano-enzyme functionalized dynamic hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Intervertebral disc degeneration (IDD) is a common chronic degenerative disease and the main pathogenic factor of low back pain in the elderly, affecting millions of people worldwide, leading to loss of mobility and reduced quality of life, and causing serious burden to the society and economy and medical care. At present, the main treatment methods for IDD in clinic are non-steroidal anti-inflammatory drug therapy and surgical treatment, which can relieve the symptoms of patients, but cannot restore the tissue structure and biomechanical function of the intervertebral disc. Nucleus pulposus is a gelatinous tissue rich in proteoglycans and type II collagen, and has a high water content, which is located in the center of the intervertebral disc and is responsible for weight-bearing, cushioning, stress absorption and maintaining elasticity, and is essential for maintaining the normal physiological activity of the intervertebral disc. Therefore, promoting the repair of nucleus pulposus is an ideal way to treat IDD.

[0003] Hydrogel, as an injectable biomaterial, has attracted widespread attention in IDD treatment due to its ability to simulate natural nucleus pulposus tissue. Hydrogel is a polymer with a three-dimensional cross-linked network structure and adjustable physicochemical properties, which exhibits many advantages in intervertebral disc repair. First, most hydrogels have good biocompatibility and low immunogenicity, and do not cause significant immune rejection. Second, the three-dimensional network structure and high water content of hydrogel provide an ideal microenvironment for cell adhesion, proliferation and survival. At the same time, bioactive molecules (such as drugs, cytokines, etc.) can be loaded in hydrogel to exert biological functions to promote intervertebral disc repair by on-demand release. In addition, by changing the material type and molecular weight, adjusting the formula and preparation method, the cross-linking degree, porosity and other parameters of hydrogel can be controlled, and thus the structure and properties of hydrogel can be adjusted to achieve the required functions and meet different application scenarios. Therefore, the diversity of hydrogel enables it to simulate the mechanical properties of natural nucleus pulposus, and most hydrogels can be implanted through minimally invasive surgery, reducing potential damage caused by surgical invasion.

[0004] For example, the prior art CN118436846A discloses a hyaluronic acid / polyaspartic acid interpenetrating network hydrogel loaded with iron-based nanoszyme and a preparation method and application thereof, 3,3'-dithiodipropionyl hydrazine is grafted onto hyaluronic acid to obtain hydrazide hyaluronic acid; aldehyde-based polyaspartic acid is synthesized by using polysuccinimide; Fe3N@Fe3O4 heterojunction nanoszyme is synthesized by using Fe3N; Fe3N@Fe3O4 heterojunction nanoszyme, hydrazide hyaluronic acid aqueous solution and aldehyde-based polyaspartic acid aqueous solution are mixed and left to stand. The heterojunction nanoszyme in the application has peroxidase and catalase activities and excellent photothermal performance, can catalyze hydrogen peroxide to generate hydroxyl radicals and oxygen, can overcome the problem of wound anoxia, can realize chemical dynamic and photothermal synergistic sterilization, and is conducive to wound healing. The hydrogel has the advantages of excellent biocompatibility, mild preparation conditions, multiple enzyme activities and strong water absorption capacity.

[0005] For example, the prior art CN118286260A discloses a manganese-based nanoszyme, a composite hydrogel and a preparation method and application thereof. The manganese-based nanoszyme is prepared by the following process: MnCl2 is dissolved in formamide to obtain a MnCl2 solution, the MnCl2 solution is heated in an oven for reaction, cooled to room temperature, the solid product is collected by centrifugation, washed and dried. The composite hydrogel mainly consists of chitosan, manganese-based nanoszyme, β-glycerophosphate sodium, hydroxyethyl cellulose and ultrapure water. The preparation method comprises the following steps: β-glycerophosphate sodium solution is added dropwise into chitosan acetic acid solution to obtain a mixed solution A; manganese-based nanoszyme is added into the mixed solution A to obtain a mixed solution B; hydroxyethyl cellulose solution is added into the mixed solution B and mixed uniformly to obtain the composite hydrogel. The composite hydrogel has important application value in the treatment of temporomandibular joint arthritis.

[0006] Excessive production of reactive oxygen species (ROS) in the nucleus pulposus is a key factor in the development of IDD, which leads to a long-term inflammatory environment and tissue structure collapse of the intervertebral disc by inducing extracellular matrix degradation and pro-inflammatory factor production. Normal nucleus pulposus tissue depends on the ability of nucleus pulposus cells to secrete extracellular matrix. However, high levels of ROS can cause cells to be in an oxidative stress state, and long-term oxidative stress eventually leads to cell death. Among them, ferroptosis is closely related to ROS accumulation, is a kind of programmed cell death dependent on iron ions and accompanied by an increase in intracellular lipid peroxides, and participates in the progression of IDD. Therefore, developing a new type of biomaterial with ROS scavenging ability, inhibiting inflammatory response and nucleus pulposus cell ferroptosis, thereby restoring cell viability and extracellular matrix balance, and promoting intervertebral disc repair, will have a significant impact on the clinical treatment of IDD. However, there is currently no such material in clinical trials for IDD treatment.

[0007] Based on the above background, the present application designs and prepares a nano-enzyme functionalized dynamic hydrogel for removing ROS in the nucleus pulposus, promoting nucleus pulposus repair, and thus realizing the recovery of the structure and function of degenerative intervertebral disc.

[0008] Currently, some biomaterials with good biomechanical properties also show good treatment prospects in the IDD field, but because they cannot target key molecules and the root cause of IDD, many have not entered clinical trials. In addition, metal substitutes for the nucleus pulposus also have the risk of stress shielding leading to adjacent vertebral disease. On the contrary, the many advantages of hydrogels make them have great application value in intervertebral disc repair. Of course, when designing hydrogels for IDD treatment, they should also meet the requirements of intervertebral disc repair for biomaterials. For example, hydrogels have certain water solubility and are easily affected by temperature, pH and other factors, so they need to be properly modified and protected to improve their stability. Because the intervertebral disc is under continuous load, and the modulus of traditional hydrogels is low and the toughness is poor, they are prone to rupture during treatment, leading to support failure, so they cannot play a long-term effective mechanical compensation function in the treatment of intervertebral discs, greatly limiting the tissue repair of intervertebral discs. In order to solve the possible problems of injectable hydrogels, the current research focus has shifted to the development of dynamic hydrogels with biological activity, simple composition and easy synthesis. Due to its reversible crosslinking characteristics, it can dissipate energy through the dissociation and recombination of dynamic bonds under external force, and play a secondary crosslinking role with related components in the extracellular matrix, synergistically enhancing the mechanical properties of hydrogels, which can act as a mechanical function compensator for the nucleus pulposus during IDD treatment and provide stable support. At the same time, through the release of biologically active molecules in response to the environment, the local microenvironment is improved and cell function is restored, thereby promoting intervertebral disc repair.

[0009] Nanocatalysts are a new type of synthetic biomaterials with natural enzyme-like catalytic activity, which have shown great application potential in the field of tissue engineering. Among them, cerium oxide (CeO x ) exhibits low cytotoxicity and high ROS scavenging efficiency in the cycling process between Ce(III) and Ce(IV), so it has attracted widespread attention in ROS-related diseases. However, the main problem faced by CeO x -based nanocatalysts in reducing ROS is that Ce(III) reacts with ROS and oxidizes to Ce(IV), limiting its catalytic activity. Therefore, it is necessary to introduce additional stabilizers to maintain the function of nanocatalysts. Currently, black phosphorus nanosheets (BPNSs, BP) have been widely studied and applied due to their unique physical and chemical properties, biocompatibility and biodegradability. However, there is no report on using BP as a stabilizer for CeO x nanocatalysts.

[0010] The present application designs and synthesizes a long-acting nanoscale enzyme targeting the nucleus pulposus, and loads it into a dynamic hydrogel, enabling it to remove ROS in the nucleus pulposus in an environmentally responsive manner; at the same time, the introduced nanoscale enzyme can serve as an energy dissipation site for the dynamic hydrogel, improving the mechanical properties of the hydrogel. Ultimately, by improving the microenvironment and restoring cell viability, the purpose of repairing the intervertebral disc is achieved. SUMMARY

[0011] The present application first provides a nanoscale enzyme functionalized dynamic hydrogel, comprising: black phosphorus nanosheets and a nanoscale enzyme.

[0012] In certain embodiments, the nanoscale enzyme is CeO x nanoscale enzyme.

[0013] In certain embodiments, the nanoscale enzyme functionalized dynamic hydrogel further comprises a cell membrane.

[0014] In certain embodiments, the cell membrane is a nucleus pulposus cell membrane.

[0015] In certain embodiments, the nanoscale enzyme functionalized dynamic hydrogel further comprises sodium tetraborate.

[0016] The present application also provides a preparation method of the above-mentioned nanoscale enzyme functionalized dynamic hydrogel, comprising the following steps:

[0017] (1) synthesis of a hydrogel precursor solution; (2) preparation of a biomimetic nanoscale enzyme; (3) preparation of a dynamic hydrogel.

[0018] In certain embodiments, the (1) synthesis of a hydrogel precursor solution comprises the following steps:

[0019] (1.1) adding 2-amino-4-hydroxy-6-methylpyrimidine to hexamethylene diisocyanate, reacting in a nitrogen environment at 100 DEG C, and after the reaction system is cooled to room temperature, adding pentanediol to the above-mentioned solution, stirring, and then collecting product 1 by suction filtration;

[0020] (1.2) after washing product 1 with pentanediol, drying the product in a vacuum oven to obtain product UPy-NCO;

[0021] (1.3) adding gelatin and UPy-NCO to dimethyl sulfoxide, reacting in a nitrogen environment at 100 DEG C, and after the reaction system is cooled to room temperature, adding pre-cooled acetone dropwise to the above-mentioned solution, stirring, and then collecting product 2 by suction filtration;

[0022] (1.4) resuspending product 2 in distilled water, dialyzing, and freeze-drying to obtain UPy-Gel.

[0023] In certain embodiments, the preparation of the (2) biomimetic nanoscale enzyme comprises the following steps:

[0024] (2.1) Preparation of BPNSs: Disperse black phosphorus powder in N-methyl-2-pyrrolidone (NMP) and ultrasonicate in the dark, centrifuge in the dark, collect the supernatant containing black phosphorus nanosheets (BPNSs), and remove the large-size black phosphorus powder; then, resuspend the BPNSs in anhydrous ethanol, centrifuge in the dark to remove residual NMP; repeat the washing with anhydrous ethanol in the dark for 3-5 times, collect the obtained BPNSs dispersed in anhydrous ethanol, and store at 4°C in the dark.

[0025] (2.2) Preparation of CeO x : Put cerium chloride heptahydrate into a flask, add oleylamine and xylene, ultrasonicate, then add distilled water, and react at 90°C for 4 hours; after the reaction is completed, add anhydrous ethanol to the flask, centrifuge for 10 minutes to wash the cerium oxide (CeO x ), repeat the operation 4 times, collect the obtained CeO x dispersed in anhydrous ethanol, and store at 4°C;

[0026] (2.3) Preparation of BP@CeO x : Mix the CeO x with the BPNSs, ultrasonicate the obtained mixture, then stir at room temperature to react; after the reaction is completed, wash the product with distilled water for 2 times, collect the obtained BP@CeO x dispersed in anhydrous ethanol, and store at 4°C.

[0027] (2.4) Preparation of MBC: Centrifuge the nucleus pulposus cell suspension, collect the precipitate; after resuspending the cell precipitate with PBS, quickly freeze the cell suspension in liquid nitrogen, then quickly heat the cell suspension to 100°C, repeat the operation 3-5 times; centrifuge the quickly frozen and thawed mixture, collect the supernatant containing cell membranes; mix the supernatant containing cell membranes with the BP@CeO x , and use a liposome extruder to repeatedly extrude the above mixture for 30-35 times to obtain the enveloped BP@CeO x (MBC).

[0028] In certain embodiments, the preparation of the (3) dynamic hydrogel comprises the following steps:

[0029] (3.1) Dissolve UPy-Gel in a PBS solution to obtain a UPy-Gel solution; at the same time, add polyvinyl alcohol (PVA) to PBS, stir overnight to obtain a 10% (w / v) PVA solution. Mix the 10% PVA solution with the 10 mg / mL UPy-Gel solution, and add a sodium tetraborate solution to form a dynamic hydrogel at room temperature, which is denoted as PG hydrogel;

[0030] (3.2) After mixing 10% PVA solution with 10 mg / mL UPy-Gel solution, BP@CeO x and MBC were added into the above PVA+UPy-Gel mixture, respectively, and the hydrogel components were fully mixed by ultrasonication, followed by the addition of sodium tetraborate solution, and the dynamic hydrogel was formed immediately at room temperature, and was recorded as PG@BC and PG@MBC hydrogel, respectively.

[0031] The application finally provides an application of the above-mentioned nano-enzyme functionalized dynamic hydrogel or the above-mentioned preparation method, and the application is one or more of a) to d);

[0032] a) for removing ROS at the nucleus site;

[0033] b) for preparing a drug for treating intervertebral disc degeneration;

[0034] c) for promoting the repair of nucleus or degenerative intervertebral disc structure and function;

[0035] d) for improving the microenvironment and restoring cell viability;

[0036] The application is for non-therapeutic purposes.

[0037] Compared with the prior art, the application has at least the following beneficial effects:

[0038] The beneficial effect of synthesis step two: the nano-enzyme based on CeO x faces a major problem in reducing ROS, that is, Ce(III) reacts with ROS and is oxidized to Ce(IV), which limits its catalytic activity. BP can maintain the Ce(III) / Ce(IV) redox cycle through self-oxidation and improve its ROS scavenging ability in the microenvironment, thereby serving as a stabilizer for CeO x nano-enzyme. In addition, MBC obtained by wrapping BP@CeO x in the nucleus cell membrane can better achieve nucleus cell targeting, so that BP@CeO x can effectively and durably scavenge intracellular ROS.

[0039] The beneficial effect of synthesis step three: the restoration of intervertebral disc structure and function requires that the material not only has a biological therapeutic effect, but also needs mechanical support. The introduction of MBC into the dynamic hydrogel can increase the energy dissipation sites of the hydrogel, thereby improving the mechanical properties of the hydrogel. At the same time, the ROS-responsive bond (borate ester bond) of the dynamic hydrogel can respond to ROS in the microenvironment, thereby delivering MBC to nucleus cells to exert an antioxidant therapeutic effect. BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 The apparent morphology of the nanoscale enzyme. Figure 1 A: TEM characterization of BP@CeO x The surface morphology of the nanoscale enzyme. Figure 1 B: Dynamic light scattering measurement. Figure 1 C: Thermal gravimetric analysis display. Figure 1 D: Zeta potential analysis of BP@CeO x .

[0041] Figure 2 Synthesis characterization of the nanoscale enzyme. Figure 2 A: FTIR spectrum of BP@CeO x showed Ce-O-Ce and Ce-O corresponding stretching vibration peaks at 444 cm-1 and 1391 cm-1, respectively. Figure 2 B: UV-Vis spectrum of BP@CeO x showed a similar wide absorption band to BPNSs and CeO x . Figure 2 C: Raman scattering spectrum was used to characterize BP, CeO x and BP@CeO x , respectively. Figure 2 D: X-ray diffraction (XRD) analyzed the physical structure of BP@CeO x .

[0042] Figure 3 Elemental analysis of the nanoscale enzyme.

[0043] Figure 4 Morphology characterization of the dynamic hydrogel. Figure 4 A: FTIR spectrum showed the characteristic peak of UPy at 1630 cm-1. Figure 4 B-D: Scanning electron microscopy results.

[0044] Figure 5 Mechanical properties of the dynamic hydrogel. Figure 5 A: Compression stress-strain curve of the original hydrogel.

[0045] Figure 5 B: Compression stress-strain curve of the hydrogel after self-healing. Figure 5 C: Compression modulus of the original PG@BC and PG@MBC hydrogels. Figure 5 D-F: Compression cycle test results. Figure 5 G-H: Rheometer was used to determine the rheological properties of PG@MBC hydrogel under different conditions.

[0046] Figure 6 Antioxidant properties of the dynamic hydrogel. Figure 6A: DPPH scavenging ability of PG@BC and PG@MBC hydrogels at each time point. Figure 6 B: H2O2 scavenging ability of PG@BC and PG@MBC hydrogels.

[0047] Figure 7 Cell viability test after co-culturing nucleus pulposus cells with hydrogels under oxidative stress for 48 hours; Figure 7 A is the staining result; Figure 7 B is the counting result.

[0048] Figure 8 Therapeutic effect of PG@MBC dynamic hydrogel after rat tail intervertebral disc puncture. Figure 8 A: Flow chart for evaluating the therapeutic effect of PG@MBC in rat tail IDD model induced by acupuncture.

[0049] Figure 8 B: X-ray and MRI examination of rat tail vertebrae, the dashed box is the experimental observation segment.

[0050] Figure 8 C: Compared with the IDD group, PG@MBC dynamic hydrogel can significantly maintain the intervertebral height, and the intervertebral height index (DHI) of this group is close to the normal control group (Con).

[0051] Figure 8 D: Calculate the water content of the intervertebral disc according to the gray value of the nucleus pulposus shown in the MRI examination results.

[0052] Figure 8 E: According to the MRI examination results, judge the Pfirrmann degeneration grade of the intervertebral disc of each group.

[0053] Figure 8 F-G: Observe the tissue structure and pathological changes of each group by hematoxylin-eosin (HE) and safranin O / fast green (SO / FG) staining. DETAILED DESCRIPTION

[0054] In order to make the technical problems, technical solutions and advantages to be solved by the present application more clear, the following will be described in detail in combination with the drawings and specific embodiments.

[0055] List of abbreviations, English and key term definitions: 1, IDD: intervertebral disc degeneration

[0056] 2, UPy-Gel: urea-based pyrimidone grafted gelatin

[0057] 3, PVA: polyvinyl alcohol

[0058] 4, BP@CeO x : black phosphorus nanosheet modified with cerium oxide

[0059] 5. MBC: BP@CeO coated with nucleus marrow cell membrane x

[0060] 6. PG: Dynamic hydrogel prepared by mixing UPy-Gel, PVA and sodium tetraborate

[0061] 7. PG@BC: PG loaded with BP@CeO x Nanoenzyme PG hydrogel

[0062] 8. PG@MBC: PG hydrogel loaded with MBC nanoenzyme

[0063] 9. ROS: Reactive oxygen species

[0064] Example 1

[0065] I. Synthesis of hydrogel precursor solution

[0066] To obtain urea-based pyrimidinone grafted gelatin (UPy-Gel), 2.5 g of 2-amino-4-hydroxy-6-methylpyrimidine was added to 22.5 mL of hexamethylene diisocyanate and reacted in a nitrogen environment at 100°C. After 16 hours of reaction, the reaction system was cooled to room temperature, 50 mL of pentanediol was added to the above solution, stirred at 200 rpm for 10 minutes, and then the product was collected by suction filtration. After washing the product with pentanediol for 3 times, the product was placed in a vacuum oven and dried for 48 hours to obtain the product UPy-NCO. 9 g of gelatin and 1 g of UPy-NCO were added to 50 mL of dimethyl sulfoxide and reacted in a nitrogen environment at 100°C. After 16 hours of reaction, the reaction system was cooled to room temperature, 300 mL of pre-cooled acetone was added dropwise to the above solution, stirred at 200 rpm for 10 minutes, and then the product was collected by suction filtration. The collected product was resuspended in distilled water and loaded into a dialysis bag with a molecular weight cut-off of 3500 Da, and dialyzed for 3 days, with distilled water changed every 6 hours. After dialysis, the product was freeze-dried to obtain UPy-Gel.

[0067] II. Preparation of biomimetic nanoenzyme

[0068] (1) Preparation of BPNSs: 50 mg of black phosphorus powder was dispersed in 50 mL of N-methyl-2-pyrrolidone (NMP) and continuously sonicated at 4°C for 20 hours at a power of 500 W in the dark. After ultrasonic treatment, the mixture was centrifuged at 2000 rpm for 10 minutes at 4°C in the dark to remove large-sized black phosphorus powder, and the supernatant containing black phosphorus nanosheets (BPNSs) was collected. Subsequently, the BPNSs were resuspended in anhydrous ethanol and centrifuged at 10000 rpm for 20 minutes at 4°C in the dark to remove residual NMP. The collected BPNSs were washed with anhydrous ethanol for 3-5 times in the dark, and then stored in anhydrous ethanol at 4°C in the dark.

[0069] (2) Preparation of CeO x : 370 mg of cerium chloride heptahydrate was put into a flask, 3.2 g of oleylamine and 15 mL of xylene were added, and ultrasonic treatment was performed for 15 minutes, followed by the addition of 1 mL of distilled water, and reaction was performed at 90°C for 4 hours. After the completion of the reaction, anhydrous ethanol was added to the flask, and centrifugation was performed at a rotation speed of 10000 rpm for 10 minutes to wash the cerium oxide (CeO x ), and this operation was repeated 4 times, and the obtained CeO x was collected and dispersed in anhydrous ethanol, and stored at 4°C.

[0070] (3) Preparation of BP@CeO x : 3 mg of CeO x was mixed with 1.2 mg of BPNSs, and the obtained mixture was subjected to ultrasonic treatment for 10 minutes, followed by stirring at room temperature for 4 hours. After the completion of the reaction, the product was washed twice with distilled water, and the obtained BP@CeO x was collected and dispersed in anhydrous ethanol, and stored at 4°C.

[0071] (4) Preparation of MBC: To prepare the encapsulated BP@CeO x as a biomimetic nanozyme, first, a liposome extruder was used to extract the cell membrane of a nucleus pulposus cell. Specifically, a nucleus pulposus cell suspension was centrifuged at a rotation speed of 5000 rpm for 15 minutes, and the precipitate was collected. After resuspending the cell precipitate with PBS (pH = 7.4), the cell suspension was rapidly frozen in liquid nitrogen, and then the cell suspension was rapidly heated to 100°C, and this operation was repeated 3-5 times. After centrifugation of the rapidly frozen and thawed mixture at a rotation speed of 10000 rpm for 20 minutes, the supernatant containing the cell membrane was collected. 1 mL of the supernatant containing the cell membrane was mixed with 0.5 mL of BP@CeO x with a concentration of 5 mg / mL, and the above 1.5 mL of mixture was repeatedly extruded 30-35 times using a liposome extruder to obtain the encapsulated BP@CeO x (MBC).

[0072] III. Preparation of a dynamic hydrogel

[0073] 10 mg of UPy-Gel was dissolved in 100 mL of PBS (pH = 7.4) to obtain a 10 mg / mL UPy-Gel solution. Simultaneously, 1 g of polyvinyl alcohol (PVA) was added to 10 mL of PBS (pH = 7.4), and the mixture was stirred overnight at 90 °C to obtain a 10% (w / v) PVA solution. 500 μL of the 10% PVA solution was mixed with 500 μL of the 10 mg / mL UPy-Gel solution, and 50 μL of a 2% sodium tetraborate solution was added. A dynamic hydrogel, denoted as PG hydrogel, formed immediately at room temperature. Similarly, after mixing 500 μL of the 10% PVA solution with 500 μL of the 10 mg / mL UPy-Gel solution, 50 μL of BP@CeO2 was added to each solution. x Add MBC to the above PVA+UPy-Gel mixture, sonicate for 10 minutes to fully mix the components of the hydrogel, then add 50 μL of 2% sodium tetraborate solution, and a dynamic hydrogel is formed immediately at room temperature, which are denoted as PG@BC and PG@MBC hydrogels, respectively.

[0074] Example 2: Phenomorphological characteristics of nanozymes

[0075] The morphological characteristics of nanozymes were identified, and the results are shown in [reference needed]. Figure 1 ; Figure 1 A: Transmission electron microscopy characterized BP@CeO x The surface morphology and corresponding energy diffraction pattern prove that in BP@CeO x Carbon (C), oxygen (O), nitrogen (N), phosphorus (P), and cerium (Ce) are evenly distributed within it. This is to enhance the absorption of BP@CeO by nucleus pulposus cells. x To improve uptake efficiency, nucleus pulposus cell membranes extracted using a liposome extruder were repeatedly extruded and coated onto BP@CeO3. x A membrane-bound nanozyme (MBC) with high homology affinity to the nucleus pulposus cell membrane was prepared. Transmission electron microscopy images showed that the MBC had good dispersibility and a uniform spherical morphology. Figure 1 B: Dynamic light scattering measurements show BP and CeO₂. x and BP@CeO x The hydrodynamic particle sizes are 192 nm, 255 nm and 459 nm, respectively. Figure 1 C: Thermogravimetric analysis shows that BP@CeO x It has thermal stability. Figure 1 D: Due to CeO x Carrying a positive charge, therefore BP@CeO is different from BP. x The zeta potential increased to -16.8 mV.

[0076] Example 3: Synthesis and Characterization of Nanozymes

[0077] Characterization of nanozyme synthesis results are shown in [link to original text]. Figure 2 ; Figure 2 A: Due to BP@CeO x The FTIR spectrum at 444 cm⁻¹ -1 and 1391cm -1 The stretching vibration peaks corresponding to Ce-O-Ce and Ce-O are shown at the respective locations. Figure 2 B: BP@CeO x It exhibits similarities to BPNSs and CeO in the ultraviolet-visible region. x Similar broad absorption bands. Figure 2 C: To investigate BP@CeO x The molecular composition of BP and CeO was characterized by Raman scattering spectroscopy. x and BP@CeO x BP@CeO x Three characteristic peaks of BP were shown, one of which was located at 363 cm⁻¹. -1 Out-of-plane mode (A) 1 g ) and located at 439cm -1 and 467cm -1 Planar mode (B) 2g and A 2 g This indicates that CeO x The modification did not change the original structure of BP. However, compared with BP alone, BP@CeO x The three characteristic peaks (A) 1 g B 2g and A 2 g They were redshifted by 2cm respectively. -1 1cm -1 and 2cm -1 This is mainly because CeO x The addition of hinders the oscillation of P atoms in the original BP, thus causing a redshift of the three Raman characteristic peaks mentioned above, and reducing the corresponding scattering energy to some extent. Figure 2 D: X-ray diffraction (XRD) analysis of BP@CeO x The physical structure of BP@CeO. x CeO appeared at 28.5°, 33.1°, 47.5° and 56.3° respectively. x The diffraction peaks (111), (200), (220), and (311) were observed, and corresponding diffraction peaks for BP (021), (040), and (060) appeared at 16.9°, 26.6°, 34.2°, and 52.4°, directly proving that BP@CeOx crystalline containing BP and CeO x .

[0078] Elemental analysis of nanoszyme

[0079] Elemental analysis of nanoszyme, see Figure 3 ; further confirmed the chemical composition of BP@CeO x by X-ray photoelectron spectroscopy (XPS). As shown in the figure, all samples appeared characteristic peaks of P atoms at 129.2 eV and 130.1 eV, characteristic peaks of PO4 3- at 133.8 eV, characteristic peaks of Ce(III) at 880.9 eV, 884.5 eV, 898.0 eV and 903.4 eV, and characteristic peaks of Ce(IV) at 888.5 eV, 907.0 eV and 916.3 eV, indicating that P atoms, PO4 3- , Ce(III) and Ce(IV) exist in all samples. In order to explore the content difference of each element in different samples, the XPS fine spectrum of BP, CeO x and BP@CeO x was used to quantitatively analyze the elements in each sample. Compared with pure CeO x , the content of Ce(III) with reducing effect in BP@CeO x increased from 43.79% to 71.21%, while the content of Ce(IV) decreased from 56.32% to 28.79%. On the contrary, compared with pure BP, the content of P atoms in BP@CeO x decreased to 16.82%, while the content of PO4 3- rose to 81.52%. It showed that the increase of reducing Ce(III) in BP@CeO x was due to the coordination of P atoms in BP with dissolved oxygen in the microenvironment to form PO4 3- , which effectively prevented Ce(III) from being oxidized to Ce(IV), laying a material foundation for greatly improving the persistence of ROS scavenging by BP@CeO x nanoszyme.

[0080] Morphological characteristics of dynamic hydrogel

[0081] Morphological characteristics of dynamic hydrogel of nanoszyme were identified, see Figure 4 ; Figure 4 A: FTIR spectrum appeared characteristic peaks of UPy at 1630 cm -1 , indicating that UPy-Gel was successfully synthesized. Figure 4B-D: The results of scanning electron microscopy showed that all dynamic hydrogels exhibited clear porous network structure, while the addition of nanoszyme led to a significant decrease in the pore size and porosity of the hydrogel interior. This may be due to the nanoszyme BP@CeO x and MBC can reduce the pore size of the hydrogel internal network structure in the form of "filler" after adding to the hydrogel, but at the same time can also improve the mechanical properties of the hydrogel as energy dissipation sites, and endow the hydrogel with the ability of shock absorption and buffering.

[0082] Example 6 Mechanical properties of dynamic hydrogels

[0083] The mechanical properties of the dynamic hydrogel of the nanoszyme were identified, and the results are shown in Figure 5 ; Figure 5 A: Compression stress-strain curve of the original hydrogel. Figure 5 B: Compression stress-strain curve of the hydrogel after self-healing. Figure 5 C: The compression modulus of the original PG@BC and PG@MBC hydrogels was 3.45 times and 3.57 times higher than that of the PG hydrogel, reaching 9.86 kPa and 10.19 kPa, respectively. The compression modulus of the PG@BC and PG@MBC hydrogels after self-healing was 7.54 kPa and 8.59 kPa, respectively, which was almost 76.49% and 84.22% of the compression modulus of the original sample. Figure 5 D-F: The results of compression cycle experiment showed that all dynamic hydrogels exhibited good fatigue resistance, which was mainly due to the fact that during the loading / unloading process, various dynamic interactions in the hydrogel effectively dissipated energy through fracture and recombination, avoiding irreversible damage to the internal network structure of the hydrogel under external force, which led to a decrease in the fatigue resistance of the material. Figure 5 G-H: The rheological properties of PG@MBC hydrogel under different conditions were determined using a rheometer. The results showed that when the shear time was extended to 5 minutes and the environmental temperature was raised from -10°C to 40°C, the storage modulus of PG@MBC hydrogel was always much higher than the dissipation modulus, proving that PG@MBC hydrogel could maintain stable elastic state under long-term shear stress and physiological environment.

[0084] Example 7 Antioxidant properties of dynamic hydrogels

[0085] The antioxidant properties of the dynamic hydrogel of the nanoszyme were identified, and the results are shown in Figure 6 ;

[0086] Experimental steps:

[0087] (1) DPPH free radical scavenging experiment: BP, CeO x , BP@CeO xand MBC were mixed with DPPH solution at 37°C in the dark environment, and the color changes of pure DPPH solution and sample and DPPH mixed solution were observed at 1st day, 4th day and 7th day, respectively. To further quantify the antioxidant capacity, the absorbance of pure DPPH solution and sample and DPPH mixed solution at 517 nm wavelength was measured by a microplate reader at a certain time point. The DPPH free radical scavenging capacity was calculated according to the following formula:

[0088]

[0089] wherein A c is the absorbance of pure DPPH solution at 517 nm, A s is the absorbance of sample and DPPH mixed solution at 517 nm.

[0090] (2) H2O2 scavenging experiment: 50 μL of BP, CeO x , BP@CeO x and MBC were mixed with 100 μM H2O2 detection reagent, and the absorbance of each group of samples at 560 nm wavelength was detected by a microplate reader after incubation at room temperature for different time at 1st day, 4th day and 7th day, respectively. The H2O2 scavenging capacity of BP@CeO x and MBC was calculated according to the standard curve.

[0091] Results description:

[0092] Figure 6 A: The DPPH scavenging capacity of PG@BC and PG@MBC hydrogels at each time point was significantly higher than that of PG hydrogel, and the DPPH scavenging capacity of the two at 7th day was 1.79 times and 1.63 times of that of PG hydrogel, respectively, indicating that the addition of nanoenzyme could significantly improve the antioxidant capacity of dynamic hydrogel.

[0093] Figure 6 B: PG@BC and PG@MBC hydrogels also showed high and persistent H2O2 scavenging capacity. After 1 day of co-culture, the residual H2O2 concentration of PG@BC and PG@MBC groups was only 8.96% and 9.48%, which was much lower than that of PG group, indicating that dynamic hydrogel could quickly scavenge H2O2. At 7th day, the residual H2O2 concentration of PG@BC group and PG@MBC group was further reduced to 1.28% and 1.34%, which proved that the H2O2 scavenging capacity of dynamic hydrogel was long-acting.

[0094] Example 8 cell activity test

[0095] The cell activity of nanoenzyme dynamic hydrogel was tested, and the results are shown in Figure 7 ; Figure 7Live / dead cell counting after adding oxidative stress inducer tert-butyl hydroperoxide (TBHP) and co-culturing with hydrogels for two days. It can be observed that the simple TBHP group has serious cell death compared with the control group, which is manifested as a large number of red staining and a decrease in green fluorescent staining. The PG@BC and PG@MBC dynamic hydrogels, due to the carrying of nano-enzymes BP@CeO x and MBC with therapeutic effect, make the cell activity recover in the action of TBHP, and the cell activity (green fluorescence) is increased to an average of 81.1% and an average of 92.9%, respectively, while the PG dynamic hydrogel, without carrying therapeutic drugs, has no statistical difference with the simple TBHP group. The above results suggest that the PG@BC or PG@MBC dynamic hydrogel can rescue its oxidative stress at the cellular level and promote cell survival.

[0096] Example 9 Rat Experiment

[0097] The therapeutic effect of PG@MBC dynamic hydrogel on rat tail intervertebral disc puncture was tested, and the results are shown in Figure 8 ;

[0098] Experimental procedure: 8-week-old rats were punctured with a 21G needle at the Co8 / 9 intervertebral disc near the tail under anesthesia, rotated 360 degrees and kept inserted for 15 seconds. The experimental group (PG@MBC) was injected with 10 μL hydrogel for treatment, and the tail vertebrae were taken for imaging examination and sectioning at 6 weeks after the operation.

[0099] Result Description:

[0100] Figure 8 A: Flow chart for evaluating the therapeutic effect of PG@MBC in rat tail IDD model induced by needle puncture.

[0101] Figure 8 B: X-ray and MRI examination of rat tail vertebrae, the dashed box is the experimental observation segment.

[0102] Figure 8 C: Compared with the IDD group, PG@MBC dynamic hydrogel can significantly maintain the intervertebral height, and the disc height index (DHI) of this group is close to the normal control group (Con).

[0103] Figure 8 D: The water content of the intervertebral disc is calculated according to the gray value of the nucleus pulposus shown in the MRI examination result, and the PG@MBC dynamic hydrogel can significantly increase the water content of the nucleus pulposus, which is similar to the Con group.

[0104] Figure 8 E: According to the MRI examination results, the Pfirrmann degeneration grade of each group of intervertebral discs is judged. The IDD group has the highest degeneration grade, while the PG@MBC group is significantly reduced.

[0105] Figure 8 F-G: The tissue structure and pathological changes of each group were observed by hematoxylin-eosin (HE) and safranin O / fast green (SO / FG) staining. The nucleus pulposus of IDD group had obvious fibrosis, and the boundary between nucleus pulposus and annulus fibrosus disappeared, and the histopathological score was significantly higher than that of Con group and PG@MBC group. On the contrary, the nucleus pulposus of PG@MBC group had normal morphology and active cells, was rich in proteoglycan, and the boundary between nucleus pulposus and annulus fibrosus was clear, and the histopathological score was close to that of Con group.

[0106] The above describes the preferred embodiments of the present application, it should be noted that for those skilled in the art, without departing from the principles of the present application, can make several improvements and refinements, these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A nano-enzyme functionalized dynamic hydrogel, characterized in that, The nano-enzyme functionalized dynamic hydrogel comprises: a urea-based pyrimidone grafted gelatin solution, a PVA solution, a black phosphorus nanosheet coated with a nucleus cell membrane and a CeO x The nano-enzyme solution and a sodium tetraborate solution are composed of: a urea-based pyrimidone grafted gelatin solution, a PVA solution, a black phosphorus nanosheet coated with a nucleus cell membrane and a CeO x Ce (III) and Ce (IV) are included in the CeO In the process of preparing the nanoenzyme functionalized dynamic hydrogel, the ureido pyrimidone grafted gelatin solution used is a ureido pyrimidone grafted gelatin solution with a concentration of 10 mg / mL, the PVA solution used has a w / v of 10%, and the sodium tetraborate solution used is a sodium tetraborate solution with a mass fraction of 2%.

2. A method for preparing a nano-enzyme functionalized dynamic hydrogel according to claim 1, characterized in that, The method comprises the following steps: (1) synthesis of a hydrogel precursor solution, the hydrogel precursor solution comprising: ureido pyrimidone grafted gelatin; (2) preparation of a biomimetic nanoenzyme; (3) preparation of a dynamic hydrogel; The (2) preparation of the biomimetic nanoenzyme comprises the following steps: (2.1) preparation of BP NSs: dispersing black phosphorus powder in N-methyl-2-pyrrolidone (NMP), ultrasonicating in the dark, centrifuging in the dark, collecting the supernatant containing black phosphorus nanosheets (BP NSs), and removing large-size black phosphorus powder; then, resuspending the BP NSs in anhydrous ethanol, centrifuging in the dark, and removing residual NMP; repeating the washing with anhydrous ethanol in the dark for 3-5 times, and collecting the obtained BP NSs, which are dispersed in anhydrous ethanol and stored in the dark at 4℃; (2.2) Preparation of CeO x : CeCI3.7H2O was taken in a flask, oleylamine and xylene were added and sonicated, followed by the addition of distilled water and reaction at 90°C for 4 hours; after completion of the reaction, anhydrous ethanol was added to the flask and the CeO x was washed by centrifugation for 10 minutes, this operation was repeated 4 times and the CeO x obtained was collected, dispersed in anhydrous ethanol and stored at 4°C; (2.3) Preparation of BP@CeO x : CeO x was mixed with BP NSs, and the obtained mixture was subjected to ultrasonic treatment, followed by stirring reaction at room temperature; after the reaction was completed, the product was washed twice with distilled water, and the obtained BP@CeO x was collected and dispersed in anhydrous ethanol, and stored at 4°C; (2.4) Preparation of MBC: The nucleus pulposus cell suspension was centrifuged to collect the precipitate; after resuspending the cell precipitate with PBS, the cell suspension was rapidly frozen in liquid nitrogen, and then rapidly heated to 100°C, and this operation was repeated 3-5 times; the rapidly frozen-thawed mixture was centrifuged to collect the supernatant containing the cell membrane; the supernatant containing the cell membrane was mixed with BP@CeO x , and the above mixture was repeatedly extruded 30-35 times using a liposome extruder to obtain the enveloped BP@CeO x , which was labeled as MBC.

3. The preparation method according to claim 2, characterized in that, The (1) synthesis of the hydrogel precursor solution comprises the following steps: (1.1) adding 2-amino-4-hydroxy-6-methylpyrimidine to hexamethylene diisocyanate, reacting in a nitrogen environment at 100℃, cooling the reaction system to room temperature, adding pentanediol to the above solution, stirring, and then collecting product 1 by suction filtration; (1.2) after washing product 1 with pentanediol, drying the product in a vacuum oven to obtain product UPy-NCO; (1.3) adding gelatin and UPy-NCO to dimethyl sulfoxide, reacting in a nitrogen environment at 100℃, cooling the reaction system to room temperature, adding pre-cooled acetone dropwise to the above solution, stirring, and then collecting product 2 by suction filtration; (1.4) resuspending product 2 in distilled water, dialyzing, and freeze-drying to obtain UPy-Gel.

4. The production method according to claim 2, characterized by, The (3) preparation of the dynamic hydrogel comprises the following steps: (3.1) dissolving UPy-Gel in a PBS solution to obtain a UPy-Gel solution; at the same time, adding polyvinyl alcohol (PVA) to PBS, stirring overnight to obtain a PVA solution with a w / v of 10%; mixing the 10% PVA solution with the 10 mg / mL UPy-Gel solution, and adding a sodium tetraborate solution, to form a dynamic hydrogel at room temperature, which is denoted as PG hydrogel; (3.2) After mixing 10% PVA solution with 10 mg / mL UPy-Gel solution, BP@CeO x and MBC were added into the above PVA+UPy-Gel mixture, respectively. The components of hydrogel were mixed thoroughly by ultrasonication, and then sodium tetraborate solution was added. The dynamic hydrogels were formed immediately at room temperature, which were recorded as PG@BC and PG@MBC hydrogels, respectively.

5. Use of the nanoenzyme functionalized dynamic hydrogel of claim 1 or the preparation method of claims 2-4 in the preparation of a drug for treating intervertebral disc degenerative disease.

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