Nano-enzyme functionalized dynamic hydrogel as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicine and tissue engineering, in particular to nano-enzyme functionalized dynamic hydrogel as well as a preparation method and application thereof. The nano-enzyme functionalized dynamic hydrogel is prepared from black phosphorus nanosheets and nano-enzyme. The preparation method of the nano-enzyme functionalized dynamic hydrogel comprises the following steps: (1) synthesizing a hydrogel precursor solution; (2) preparing the bionic nano enzyme; and (3) preparing the dynamic hydrogel. According to the invention, BP can maintain Ce (III) / Ce (IV) redox cycle through autooxidation and improve the ROS removal capability of BP in a microenvironment, so that BP is used as a stabilizer of CeOx nano-enzyme. Besides, the BP (at) CeOx is wrapped in a nucleus pulposus cell membrane to obtain MBC, so that nucleus pulposus cell targeting can be better realized, and intracellular ROS can be durably and effectively removed by the BP (at) CeOx.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and tissue engineering, and in particular to a nanozyme functionalized dynamic hydrogel and a preparation method and application thereof. Background Art

[0002] Intervertebral disc degenerative disease (IDD) is a common chronic degenerative disease and the main cause of low back pain in the elderly. It affects millions of people worldwide, leading to loss of mobility and reduced quality of life, and placing a serious burden on social economy and health care. At present, the main clinical treatments for IDD are non-steroidal anti-inflammatory drugs and surgical treatment. Although they can relieve the patient's symptoms, they cannot restore the tissue structure and biomechanical function of the intervertebral disc. The nucleus pulposus is a gel-like tissue rich in proteoglycans and type II collagen and high in water content. It is located in the center of the intervertebral disc and is responsible for load-bearing, buffering, absorbing stress, and maintaining elasticity. It is essential to maintain the normal physiological activities of the intervertebral disc. Therefore, promoting nucleus pulposus repair is an ideal way to treat IDD.

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

[0004] For example, prior art CN118436846A discloses a hyaluronic acid / polyaspartic acid interconnected network hydrogel loaded with iron-based nanozymes and its preparation method and application, 3,3'-dithiodipropionyl hydrazide is grafted on hyaluronic acid to obtain hydrazide hyaluronic acid; polysuccinimide is used to synthesize aldehyde-modified polyaspartic acid; Fe3N is used to synthesize Fe3N@Fe3O4 heterojunction nanozyme; Fe3N@Fe3O4 heterojunction nanozyme, hydrazide hyaluronic acid aqueous solution, aldehyde-modified polyaspartic acid aqueous solution are mixed and allowed to stand. In the present invention, heterojunction nanozyme has the activity of peroxidase and catalase and excellent photothermal performance, can catalyze hydrogen peroxide to generate hydroxyl radicals and oxygen, can overcome the problem of wound hypoxia, and can achieve chemical power and photothermal synergistic sterilization, which is beneficial to wound healing. The hydrogel has the advantages of excellent biocompatibility, mild preparation conditions, multiple enzyme activity and strong water absorption capacity.

[0005] For example, the prior art CN118286260A discloses a manganese-based nanozyme, a composite hydrogel, and a preparation method and application thereof, wherein the manganese-based nanozyme is prepared by the following process: dissolving MnCl2 in formamide to obtain a MnCl2 solution, heating the MnCl2 solution in an oven for reaction, cooling to room temperature, centrifuging to collect the solid product, washing, and drying. The composite hydrogel is mainly composed of chitosan, manganese-based nanozyme, sodium β-glycerophosphate, hydroxyethyl cellulose, and ultrapure water, and the preparation method comprises the following steps: adding sodium β-glycerophosphate solution dropwise to chitosan acetic acid solution to obtain a mixed solution A; adding manganese-based nanozyme to the mixed solution A to obtain a mixed solution B; adding hydroxyethyl cellulose solution to the mixed solution B, and mixing evenly to obtain. The composite hydrogel provided by the present invention has important application value in the treatment of temporomandibular osteoarthritis.

[0006] The overproduction of reactive oxygen species (ROS) in the nucleus pulposus is a key factor in the development of IDD. By inducing extracellular matrix degradation and the production of proinflammatory factors, the intervertebral disc is in an inflammatory environment for a long time and the tissue structure collapses. 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 a state of oxidative stress, and long-term oxidative stress eventually leads to cell death. Among them, ferroptosis is closely related to ROS accumulation. It is a type of programmed cell death that is iron-dependent and accompanied by increased intracellular lipid peroxides, and is involved in the progression of IDD. Therefore, the development of new biomaterials with ROS scavenging capabilities will have a significant impact on the clinical treatment of IDD by inhibiting inflammatory responses and ferroptosis of nucleus pulposus cells, thereby restoring cell viability and extracellular matrix balance and promoting intervertebral disc repair. However, no such materials have yet entered clinical trials for the treatment of IDD.

[0007] Based on the above background, the present invention designs and prepares a nanozyme functionalized dynamic hydrogel for removing ROS in the nucleus pulposus, promoting nucleus pulposus repair, and thereby achieving the restoration of the structure and function of the degenerated intervertebral disc.

[0008] At present, some biomaterials with good biomechanical properties have also shown good therapeutic prospects in the field of IDD, but many of them have not entered clinical trials because they cannot target the key molecules of degeneration and the root causes of IDD. In addition, metal substitutes for nucleus pulposus also have the risk of stress shielding leading to adjacent vertebral disease. On the contrary, the many advantages of hydrogels make them of great application value in intervertebral disc repair. Of course, when designing hydrogels for IDD treatment, the requirements of intervertebral disc repair for biomaterials should also be met. For example, hydrogels have a 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 traditional hydrogels have a low modulus and poor toughness, they are prone to rupture during treatment, resulting in support failure. Therefore, they cannot play a long-term and effective mechanical compensation function during the treatment of intervertebral discs, which greatly limits 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 that are bioactive, simple in composition and easy to synthesize. Due to its reversible cross-linking properties, it dissipates energy through the dissociation and recombination of dynamic bonds under the action of external force, and exerts secondary cross-linking with related components in the extracellular matrix to synergistically enhance the mechanical properties of the hydrogel. During the treatment of IDD, it can serve as a mechanical function compensator of the nucleus pulposus and play a stable supporting role. At the same time, it releases bioactive molecules through environmental responsiveness, improves the local microenvironment and restores cell function, thereby promoting intervertebral disc repair.

[0009] Nanozymes are a new type of synthetic biomaterial with natural enzyme-like catalytic activity, showing great application potential in the field of tissue engineering. x ) exhibits low cytotoxicity and high ROS scavenging efficiency during the cycle between Ce(III) and Ce(IV), and thus has attracted extensive attention in ROS-related diseases. x Nanozymes face a major problem in ROS reduction applications, namely, Ce(III) reacts with ROS and oxidizes to Ce(IV), limiting their catalytic activity. Therefore, it is necessary to introduce additional stabilizers to maintain the function of nanozymes. Currently, black phosphorus nanosheets (BPNSs, BP) have been widely studied and applied due to their unique physicochemical properties, biocompatibility, and biodegradability. However, the use of BP as CeO x There are no reports on stabilizers for nanozymes.

[0010] The present invention will design and synthesize a long-acting nanozyme targeting the nucleus pulposus, and load it into the dynamic hydrogel, so that it can remove ROS in the nucleus pulposus in an environmentally responsive manner; at the same time, the introduced nanozyme can serve as an energy dissipation site of the dynamic hydrogel to improve the mechanical properties of the hydrogel. Ultimately, the purpose of repairing the intervertebral disc is achieved by improving the microenvironment and restoring cell vitality. Summary of the invention

[0011] The present invention first provides a nanozyme functionalized dynamic hydrogel, wherein the nanozyme functionalized dynamic hydrogel comprises: black phosphorus nanosheets and nanozymes.

[0012] In certain embodiments, the nanozyme is CeO x Nanozymes.

[0013] In certain embodiments, the nanozyme-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 nanozyme functionalized dynamic hydrogel further comprises sodium tetraborate.

[0016] The present invention also provides a method for preparing the above-mentioned nanozyme functionalized dynamic hydrogel, the method comprising the following steps:

[0017] (1) Synthesis of hydrogel precursor solution; (2) Preparation of biomimetic nanozymes; (3) Preparation of dynamic hydrogels.

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

[0019] (1.1) Add 2-amino-4-hydroxy-6-methylpyrimidine to hexamethylene diisocyanate and react in a nitrogen environment at 100° C. After the reaction system is cooled to room temperature, pentanediol is added to the above solution, stirred, and then the product 1 is collected by suction filtration;

[0020] (1.2) After washing the product 1 with pentanediol, the product was placed in a vacuum oven and dried to obtain the product UPy-NCO;

[0021] (1.3) Add gelatin and UPy-NCO to dimethyl sulfoxide and react in a nitrogen environment at 100°C. After the reaction system is cooled to room temperature, add pre-cooled acetone dropwise to the above solution, stir, and then collect the product 2 by suction filtration;

[0022] (1.4) The product 2 was resuspended in distilled water, dialyzed, and freeze-dried to obtain UPy-Gel.

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

[0024] (2.1) Preparation of BPNSs: black phosphorus powder was dispersed in N-methyl-2-pyrrolidone (NMP), sonicated in the dark, centrifuged in the dark, and the supernatant containing black phosphorus nanosheets (BPNSs) was collected to remove large-sized black phosphorus powder; then, the BPNSs were resuspended in anhydrous ethanol and centrifuged in the dark to remove residual NMP; the anhydrous ethanol was used for repeated washing 3-5 times in the dark, and the collected BPNSs were dispersed in anhydrous ethanol and stored in the dark at 4°C.

[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 this operation 4 times, and collect the obtained CeO x Dispersed in anhydrous ethanol and stored at 4°C;

[0026] (2.3) Preparation of BP@CeO x :CeO x The mixture was mixed with BPNSs, and then the obtained mixture was subjected to ultrasonic treatment and then stirred at room temperature for reaction. After the reaction was completed, the product was washed twice with distilled water and the obtained BP@CeO x Disperse in anhydrous ethanol and store at 4°C.

[0027] (2.4) Preparation of MBC: The nucleus pulposus cell suspension was centrifuged and the precipitate was collected; after the cell precipitate was resuspended in PBS, the cell suspension was placed in liquid nitrogen for rapid freezing, and then the cell suspension was rapidly heated to 100°C, and this operation was repeated 3-5 times; the rapid freeze-thaw mixture was centrifuged and the supernatant containing the cell membrane was collected; the supernatant containing the cell membrane was mixed with BP@CeO x Mix and extrude the mixture 30-35 times using a liposome extruder to obtain the coated BP@CeO x (MBC).

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

[0029] (3.1) UPy-Gel was dissolved in PBS solution to obtain UPy-Gel solution; polyvinyl alcohol (PVA) was added to PBS and stirred overnight to obtain 10% (w / v) PVA solution. The 10% PVA solution was mixed with the 10 mg / mL UPy-Gel solution and sodium tetraborate solution was added to form a dynamic hydrogel at room temperature, which was recorded 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 to the PVA+UPy-Gel mixture, and ultrasound was used to fully mix the components of the hydrogel. Then, sodium tetraborate solution was added to form dynamic hydrogels immediately at room temperature, which were recorded as PG@BC and PG@MBC hydrogels, respectively.

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

[0032] a) Used to remove ROS in the nucleus pulposus;

[0033] b) preparing a medicament for treating intervertebral disc degenerative diseases;

[0034] c) Used to promote the structural and functional repair of the nucleus pulposus or degenerative intervertebral disc;

[0035] d) Used to improve microenvironment and restore cell vitality;

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

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

[0038] Beneficial effects of the second step of synthesis: based on CeO x Nanozymes face a major problem in ROS reduction applications, namely, Ce(III) reacts with ROS and oxidizes to Ce(IV), limiting its catalytic activity. BP can maintain the Ce(III) / Ce(IV) redox cycle through auto-oxidation and improve its ROS scavenging ability in the microenvironment, thus acting as a CeO x Nanozyme stabilizer. In addition, BP@CeO x The MBC encapsulated in the nucleus pulposus cell membrane can better achieve the targeting of nucleus pulposus cells, making BP@CeO x It can effectively and persistently remove intracellular ROS.

[0039] Beneficial effects of synthesis step three: The restoration of intervertebral disc structure and function requires materials that not only have biological therapeutic effects, but also mechanical support. Introducing MBC into dynamic hydrogels can increase the energy dissipation sites of hydrogels, thereby improving the mechanical properties of hydrogels. At the same time, the ROS-responsive bonds (boronate bonds) of dynamic hydrogels can respond to ROS in the microenvironment, thereby delivering MBC to nucleus pulposus cells to exert antioxidant therapeutic effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Surface morphology characteristics of nanozymes. Figure 1 A: Transmission electron microscopy characterization of BP@CeO x Surface morphology diagram. Figure 1 B: Dynamic light scattering measurement graph. Figure 1 C: Thermogravimetric analysis display. Figure 1 D: BP@CeO x Zeta potential analysis diagram.

[0041] Figure 2 Synthesis and characterization of nanozymes. Figure 2 A: BP@CeO x The FTIR spectrum showed stretching vibration peaks corresponding to Ce-O-Ce and Ce-O at 444cm-1 and 1391cm-1, respectively. Figure 2 B: BP@CeO x In the UV-visible region, it shows the same x Similar broad absorption bands. Figure 2 C: Raman scattering spectroscopy was used to characterize BP and CeO x and BP@CeO x . Figure 2 D: X-ray diffraction (XRD) analysis of BP@CeO x physical structure.

[0042] Figure 3 Elemental analysis diagram of nanozyme.

[0043] Figure 4 Morphological characteristics of dynamic hydrogels. Figure 4 A: The FTIR spectrum shows a characteristic peak of UPy at 1630cm-1. Figure 4 BD: SEM results.

[0044] Figure 5 Graph of the mechanical properties of dynamic hydrogels. Figure 5 A: Compressive stress-strain curve of the original hydrogel.

[0045] Figure 5 B: Compressive stress-strain curve of the hydrogel after self-healing. Figure 5 C: Compression modulus of original PG@BC and PG@MBC hydrogels. Figure 5 DF: Compression cycle test results. Figure 5 GH: The rheological properties of PG@MBC hydrogel under different conditions were determined using a rheometer.

[0046] Figure 6 Antioxidant properties of dynamic hydrogels. 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 activity test after 48 hours of co-culture of nucleus pulposus cells and hydrogel under oxidative stress induction; 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 coccygeal disc puncture in rats. Figure 8 A: Flowchart for evaluating the therapeutic effect of PG@MBC in acupuncture-induced rat coccygeal IDD model.

[0049] Figure 8 B: X-ray and MRI examination of the rat's coccygeal vertebrae; the dotted box is the experimental observation segment.

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

[0051] Figure 8 D: The water content of the intervertebral disc is calculated based on the gray value of the nucleus pulposus shown in the MRI examination results.

[0052] Figure 8 E: Based on the MRI examination results, the Pfirrmann degeneration grade of the intervertebral disc in each group was determined.

[0053] Figure 8 FG: The tissue structure and pathological changes of each group were observed by hematoxylin and eosin (HE) and safranin 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 invention more clear, a detailed description will be given below with reference to the accompanying drawings and specific embodiments.

[0055] List of abbreviations, English and key term definitions: 1. IDD: Intervertebral Disc Degeneration

[0056] 2. UPy-Gel: ureidopyrimidone grafted gelatin

[0057] 3. PVA: Polyvinyl alcohol

[0058] 4. BP@CeO x :Cerium oxide modified black phosphorus nanosheets

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

[0060] 6. PG: Dynamic hydrogel made of UPy-Gel, PVA and sodium tetraborate

[0061] 7. PG@BC: Equipped with BP@CeO x Nanozyme PG hydrogel

[0062] 8. PG@MBC: PG hydrogel with MBC nanozymes

[0063] 9. ROS: Reactive oxygen species

[0064] Example 1

[0065] 1. Synthesis of hydrogel precursor solution

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

[0067] 2. Preparation of biomimetic nanozymes

[0068] (1) Preparation of BPNSs: Disperse 50 mg of black phosphorus powder in 50 mL of N-methyl-2-pyrrolidone (NMP) and continuously sonicate at 500 W for 20 hours at 4°C in the dark. After the sonication, centrifuge the mixture at 2000 rpm at 4°C in the dark for 10 minutes, collect the supernatant containing black phosphorus nanosheets (BPNSs), and remove large-sized black phosphorus powder. Subsequently, resuspend the BPNSs in anhydrous ethanol and centrifuge at 10000 rpm at 4°C in the dark for 20 minutes to remove residual NMP. Repeat the washing with anhydrous ethanol in the dark for 3-5 times, and the collected BPNSs are dispersed in anhydrous ethanol and stored at 4°C in the dark.

[0069] (2) Preparation of CeO x : 370 mg of cerium chloride heptahydrate was placed in a flask, 3.2 g of oleylamine and 15 mL of xylene were added, and ultrasonic treatment was performed for 15 minutes, followed by addition of 1 mL of distilled water, and the reaction was carried out at 90°C for 4 hours. After the reaction was completed, anhydrous ethanol was added to the flask, and the cerium oxide (CeO) was washed by centrifugation at 10,000 rpm for 10 minutes. x ), repeat this operation 4 times, and collect the obtained CeO x Disperse in anhydrous ethanol and store at 4°C.

[0070] (3) Preparation of BP@CeO x : 3 mg CeO x The mixture was mixed with 1.2 mg BPNSs, and the resulting mixture was ultrasonically treated for 10 minutes, and then stirred at room temperature for 4 hours. After the reaction was completed, the product was washed twice with distilled water, and the obtained BP@CeO x Disperse in anhydrous ethanol and store at 4°C.

[0071] (4) Preparation of MBC: To prepare the coated BP@CeO x As a biomimetic nanozyme, the nucleus pulposus cell membrane is first extracted using a liposome extruder. Specifically, the nucleus pulposus cell suspension is centrifuged at 5000 rpm for 15 minutes, and the precipitate is collected. After resuspending the cell precipitate with PBS (pH = 7.4), the cell suspension is placed in liquid nitrogen for rapid freezing, and then the cell suspension is rapidly heated to 100 ° C, and this operation is repeated 3-5 times. After the rapid freeze-thaw mixture is centrifuged at 10000 rpm for 20 minutes, the supernatant containing the cell membrane is collected. 1 mL of the supernatant containing the cell membrane is mixed with 0.5 mL of BP@CeO at a concentration of 5 mg / mL. x Mix and repeatedly extrude the above 1.5 mL mixed solution 30-35 times using a liposome extruder to obtain the coated BP@CeO x (MBC).

[0072] 3. Preparation of dynamic hydrogel

[0073] 10 mg UPy-Gel was dissolved in 100 mL PBS (pH = 7.4) solution to obtain a UPy-Gel solution with a concentration of 10 mg / mL. At the same time, 1 g polyvinyl alcohol (PVA) was added to 10 mL PBS (pH = 7.4) and stirred overnight at 90 ° C to obtain a 10% (w / v) PVA solution. 500 μL of 10% PVA solution was mixed with 500 μL of 10 mg / mL UPy-Gel solution, and 50 μL of 2% sodium tetraborate solution was added to form a dynamic hydrogel at room temperature, which was recorded as PG hydrogel. Similarly, after 500 μL of 10% PVA solution was mixed with 500 μL of 10 mg / mL UPy-Gel solution, 50 μL of BP@CeO x and MBC were added to the PVA+UPy-Gel mixture, and ultrasonic treatment was performed for 10 minutes to fully mix the components of the hydrogel. Subsequently, 50 μL of 2% sodium tetraborate solution was added to form dynamic hydrogels immediately at room temperature, which were recorded as PG@BC and PG@MBC hydrogels, respectively.

[0074] Example 2: Apparent morphological characteristics of nanozymes

[0075] The surface morphology of the nanozyme was identified, and the results are shown in Figure 1 ; Figure 1 A: Transmission electron microscopy characterization of BP@CeO x The corresponding energy diffraction spectrum image proves that in BP@CeO x Carbon (C), oxygen (O), nitrogen (N), phosphorus (P) and cerium (Ce) are evenly distributed in the nucleus pulposus cells. x The uptake efficiency was improved by repeatedly extruding the nucleus pulposus cell membrane extracted by liposome extruder and encapsulating it on BP@CeO x On the surface, a coated nanozyme (MBC) with high homology affinity to the nucleus pulposus cell membrane was prepared. Transmission electron microscopy images showed that MBC had good dispersibility and uniform spherical morphology. Figure 1 B: Dynamic light scattering measurement shows that BP, CeO x and BP@CeO x The hydrodynamic particle sizes are 192nm, 255nm and 459nm respectively. Figure 1 C: Thermogravimetric analysis shows that BP@CeO x Thermally stable. Figure 1 D: Due to CeO x Positively charged, BP@CeO x The Zeta potential increased to -16.8 mV.

[0076] Example 3 Synthesis and Characterization of Nanozymes

[0077] The synthesis and characterization of nanozymes are shown in Figure 2 ; Figure 2 A: Due to BP@CeO x FTIR spectrum at 444 cm -1 and 1391cm -1 The stretching vibration peaks corresponding to Ce-O-Ce and Ce-O are shown respectively. Figure 2 B: BP@CeO x In the UV-visible region, it shows the same x Similar broad absorption bands. Figure 2 C: To explore BP@CeO x The molecular composition of BP and CeO was characterized by Raman scattering spectroscopy. x and BP@CeO x BP@CeO x It shows three characteristic peaks of BP, which are located at 363cm -1 Out-of-plane mode (A 1 g ) and located at 439cm -1 and 467cm -1 Planar mode (B 2g and A 2 g ), indicating that CeO x The modification did not cause the change of the original structure of BP. However, compared with pure BP, BP@CeO x The three characteristic peaks (A 1 g , B 2g and A 2 g ) were red-shifted by 2 cm -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 the above three Raman characteristic peaks to red-shift and reducing the corresponding scattering energy to a certain extent. Figure 2 D: X-ray diffraction (XRD) analysis of BP@CeO x The physical structure of BP@CeO x The corresponding CeO appeared at 28.5°, 33.1°, 47.5° and 56.3° respectively. x The diffraction peaks of (111), (200), (220) and (311) appeared at 16.9°, 26.6°, 34.2° and 52.4°, which corresponded to the diffraction peaks of BP (021), (040) and (060), which directly proved that BP@CeOx Contains BP and CeO x The crystal.

[0078] Example 4 Elemental Analysis of Nanozymes

[0079] Elemental analysis of nanozymes, results are shown in Figure 3 ; X-ray photoelectron spectroscopy (XPS) was used to further confirm that BP@CeO x As shown in the figure, all samples have characteristic peaks of P atoms at 129.2eV and 130.1eV, and PO4 at 133.8eV. 3- The characteristic peaks of Ce(III) appeared at 880.9eV, 884.5eV, 898.0eV and 903.4eV, and the characteristic peaks of Ce(IV) appeared at 888.5eV, 907.0eV and 916.3eV, indicating that P atoms, PO4 3- , Ce(III) and Ce(IV). In order to explore the content differences of each element in different samples, BP, CeO x and BP@CeO x XPS fine spectrum was used to quantitatively analyze the elements in each sample. x In comparison, BP@CeO x The content of Ce(III) with reducing effect in BP@CeO increased from 43.79% to 71.21%, while the content of Ce(IV) decreased from 56.32% to 28.79%. x The P atomic content in the PO4 3- It increased to 81.52%. x The increase in the reducing Ce(III) is due to the phosphorus atom in BP coordinating with dissolved oxygen in the microenvironment to form PO4 3- , thus effectively preventing Ce(III) from being oxidized to Ce(IV), which greatly improves the BP@CeO x The persistence of nanozymes in clearing ROS lays the material foundation.

[0080] Example 5 Morphological characteristics of dynamic hydrogel

[0081] The morphological characteristics of the dynamic hydrogel of the nanozyme were identified, and the results are shown in Figure 4 ; Figure 4 A: FTIR spectrum at 1630cm -1 The characteristic peak of UPy appeared at , indicating that UPy-Gel was successfully synthesized. Figure 4BD: Scanning electron microscopy results show that all dynamic hydrogels exhibit a clear porous network structure, and the addition of nanozymes leads to a significant decrease in the internal pore size and porosity of the hydrogels. This may be due to the nanozyme BP@CeO x After adding MBC to hydrogel, it can reduce the pore size of the internal network structure of the hydrogel in the form of "filler", but it may also serve as an energy dissipation site to improve the mechanical properties of the hydrogel and give the hydrogel the ability to buffer and reduce shock.

[0082] Example 6 Mechanical properties of dynamic hydrogels

[0083] The mechanical properties of the nanozyme dynamic hydrogel were identified, and the results are shown in Figure 5 ; Figure 5 A: Compressive stress-strain curve of the original hydrogel. Figure 5 B: Compressive stress-strain curve of the hydrogel after self-healing. Figure 5 C: The compression moduli of the original PG@BC and PG@MBC hydrogels are 3.45 and 3.57 times higher than that of the PG hydrogel, reaching 9.86 kPa and 10.19 kPa, respectively. The compression moduli of the self-healed PG@BC and PG@MBC hydrogels are 7.54 kPa and 8.59 kPa, respectively, almost reaching 76.49% and 84.22% of the compression modulus of the original samples. Figure 5 DF: The results of compression cycle experiments show that all dynamic hydrogels exhibit good anti-fatigue properties. This is mainly because during the loading / unloading process, the various dynamic interactions in the hydrogel effectively dissipate energy through fracture and recombination, avoiding irreversible damage to the internal network structure of the hydrogel under the action of external force, which leads to a decrease in the material's anti-fatigue performance. Figure 5 GH: The rheological properties of PG@MBC hydrogel under different conditions were measured using a rheometer. The results showed that when the shear time was extended to 5 minutes and the ambient temperature increased 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 can maintain a 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 nanozyme dynamic hydrogel 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 xThe DPPH solution and MBC were fully mixed with the DPPH solution in a dark environment at 37°C, and the color changes of the pure DPPH solution and the sample-DPPH mixture were observed on the 1st, 4th and 7th days. To further quantify the antioxidant capacity, the absorbance of the pure DPPH solution and the sample-DPPH mixture at a wavelength of 517nm was measured using an ELISA instrument at specific time points. The DPPH free radical scavenging capacity was calculated according to the following formula:

[0088]

[0089] Among them, A c is the absorbance of pure DPPH solution at 517nm, A s It is the absorbance of the mixture of sample and DPPH at 517nm.

[0090] (2) H2O2 removal experiment: 50 μL BP, CeO x 、BP@CeO x and MBC were mixed with 100 μM H2O2 detection reagent, respectively, and incubated at room temperature for different time periods. The absorbance of each group of samples at 560 nm was measured by an ELISA instrument on the 1st, 4th and 7th day. x and the ability of MBC to scavenge H2O2.

[0091] Result description:

[0092] Figure 6 A: The DPPH scavenging abilities of PG@BC and PG@MBC hydrogels at each time point were significantly higher than those of PG hydrogels, and on the 7th day, their DPPH scavenging abilities were 1.79 times and 1.63 times that of PG hydrogels, respectively, indicating that the addition of nanozymes can significantly improve the antioxidant capacity of dynamic hydrogels.

[0093] Figure 6 B: PG@BC and PG@MBC hydrogels also showed efficient and long-lasting H2O2 scavenging ability. After 1 day of co-culture, the concentration of remaining H2O2 in the PG@BC and PG@MBC groups was only 8.96% and 9.48%, which was much lower than that in the PG group, indicating that the dynamic hydrogel can quickly remove H2O2. On the 7th day, the concentration of remaining H2O2 in the PG@BC and PG@MBC groups further decreased to 1.28% and 1.34%, proving that the ability of dynamic hydrogels to remove H2O2 is long-lasting.

[0094] Example 8 Cell activity test

[0095] The cell activity of the nanozyme dynamic hydrogel was tested, and the results are shown in Figure 7 ; Figure 7The live / dead cell counts after two days of co-culture with hydrogels after adding tert-butyl hydroperoxide (TBHP), an oxidative stress inducer. It can be observed that the TBHP group alone showed severe cell death compared with the control group, as shown by the appearance of a large amount of red staining and the reduction of green fluorescence staining. The PG@BC and PG@MBC dynamic hydrogels were loaded with the therapeutic nanozyme BP@CeO x The PG@BC and MBC dynamic hydrogels restored cell activity under the action of TBHP, increasing cell activity (green fluorescence) to an average of 81.1% and 92.9%, respectively. However, since the PG dynamic hydrogels did not carry any therapeutic drugs, there was no statistical difference between them and the TBHP group alone. The above results suggest that PG@BC or PG@MBC dynamic hydrogels can rescue oxidative stress at the cellular level and promote cell survival.

[0096] Example 9 Rat Experiment

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

[0098] Experimental procedures: 8-week-old rats were anesthetized and the proximal Co8 / 9 disc of the tail was punctured with a 21G needle, rotated 360 degrees and kept inserted for 15 seconds. The experimental group (PG@MBC) was injected with 10μL of hydrogel for treatment, and the coccyx was removed for imaging examination and sectioning at 6 weeks after surgery.

[0099] Result description:

[0100] Figure 8 A: Flowchart for evaluating the therapeutic effect of PG@MBC in acupuncture-induced rat coccygeal IDD model.

[0101] Figure 8 B: X-ray and MRI examination of the rat's coccygeal vertebrae; the dotted box is the experimental observation segment.

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

[0103] Figure 8 D: The water content of the intervertebral disc was calculated based on the grayscale value of the nucleus pulposus shown in the MRI examination results. PG@MBC dynamic hydrogel can significantly increase the water content of the nucleus pulposus, which is similar to that of the Con group.

[0104] Figure 8 E: Based on the MRI results, the Pfirrmann degeneration grade of each group's intervertebral disc was determined. The IDD group had the highest degeneration grade, while the PG@MBC group had a significantly lower grade.

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

[0106] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A nanozyme functionalized dynamic hydrogel, characterized in that: The nanozyme functionalized dynamic hydrogel comprises: black phosphorus nanosheets and nanozymes.

2. The nanozyme functionalized dynamic hydrogel according to claim 1, characterized in that: The nanozyme is CeO x Nanozymes.

3. The nanozyme functionalized dynamic hydrogel according to claim 1, characterized in that: The nanozyme functionalized dynamic hydrogel also includes a cell membrane.

4. The nanozyme functionalized dynamic hydrogel according to claim 3, characterized in that: The cell membrane is a nucleus pulposus cell membrane.

5. The nanozyme functionalized dynamic hydrogel according to claim 1, characterized in that: The nanozyme functionalized dynamic hydrogel also includes sodium tetraborate.

6. A method for preparing the nanozyme functionalized dynamic hydrogel according to claim 1, characterized in that: The method comprises the following steps: (1) Synthesis of hydrogel precursor solution; (2) Preparation of biomimetic nanozymes; (3) Preparation of dynamic hydrogels.

7. The preparation method according to claim 6, characterized in that: The synthesis of the (1) hydrogel precursor solution comprises the following steps: (1.1) Add 2-amino-4-hydroxy-6-methylpyrimidine to hexamethylene diisocyanate and react in a nitrogen environment at 100° C. After the reaction system is cooled to room temperature, pentanediol is added to the above solution, stirred, and then the product 1 is collected by suction filtration; (1.2) After washing the product 1 with pentanediol, the product was placed in a vacuum oven and dried to obtain the product UPy-NCO; (1.3) Add gelatin and UPy-NCO to dimethyl sulfoxide and react in a nitrogen environment at 100°C. After the reaction system is cooled to room temperature, add pre-cooled acetone dropwise to the above solution, stir, and then collect the product 2 by suction filtration; (1.4) The product 2 was resuspended in distilled water, dialyzed, and freeze-dried to obtain UPy-Gel.

8. The preparation method according to claim 6, characterized in that: The preparation of the (2) bionic nanozyme comprises the following steps: (2.1) Preparation of BP NSs: black phosphorus powder was dispersed in N-methyl-2-pyrrolidone (NMP), sonicated in the dark, centrifuged in the dark, and the supernatant containing black phosphorus nanosheets (BP NSs) was collected to remove large-sized black phosphorus powder; then, BP NSs were resuspended in anhydrous ethanol and centrifuged in the dark to remove residual NMP; the anhydrous ethanol was repeatedly used for washing in the dark for 3-5 times, and the collected BP NSs were dispersed in anhydrous ethanol and stored in the dark at 4°C; (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 this operation 4 times, and collect the obtained CeO x Dispersed in anhydrous ethanol and stored at 4°C; (2.3) Preparation of BP@CeO x :CeO x The mixture was mixed with BP NSs, and then the obtained mixture was subjected to ultrasonic treatment and then stirred at room temperature for reaction. After the reaction was completed, the product was washed twice with distilled water and the obtained BP@CeO x Dispersed in anhydrous ethanol and stored at 4°C; (2.4) Preparation of MBC: The nucleus pulposus cell suspension was centrifuged and the precipitate was collected; after the cell precipitate was resuspended in PBS, the cell suspension was placed in liquid nitrogen for rapid freezing, and then the cell suspension was rapidly heated to 100°C, and this operation was repeated 3-5 times; the rapid freeze-thaw mixture was centrifuged and the supernatant containing the cell membrane was collected; the supernatant containing the cell membrane was mixed with BP@CeO x Mix and extrude the mixture 30-35 times using a liposome extruder to obtain the coated BP@CeO x (MBC).

9. The preparation method according to claim 6, characterized in that: The preparation of the (3) dynamic hydrogel comprises the following steps: (3.1) UPy-Gel was dissolved in PBS solution to obtain UPy-Gel solution; polyvinyl alcohol (PVA) was added to PBS and stirred overnight to obtain 10% (w / v) PVA solution; the 10% PVA solution was mixed with 10 mg / mL UPy-Gel solution, and sodium tetraborate solution was added to form a dynamic hydrogel at room temperature, which was recorded as PG hydrogel; (3.2) After mixing 10% PVA solution with 10 mg / mL UPy-Gel solution, BP@CeO x and MBC were added to the PVA+UPy-Gel mixture, and ultrasound was used to fully mix the components of the hydrogel. Then, sodium tetraborate solution was added to form dynamic hydrogels immediately at room temperature, which were recorded as PG@BC and PG@MBC hydrogels, respectively.

10. An application of the nanozyme functionalized dynamic hydrogel according to any one of claims 1 to 5 or the preparation method according to claims 6 to 9, characterized in that: The application is one or more of a) to d); a) Used to remove ROS in the nucleus pulposus; b) preparing a medicament for treating intervertebral disc degenerative diseases; c) Used to promote the structural and functional repair of the nucleus pulposus or degenerative intervertebral disc; d) Used to improve microenvironment and restore cell vitality; The use is for non-therapeutic purposes.

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