An injectable antioxidant hydrogel adapted to the mechanical properties of the nucleus pulposus, its preparation method and application

Abstract

This invention relates to the field of medical technology, specifically to an injectable antioxidant hydrogel adapted to the mechanical properties of the nucleus pulposus, its preparation method, and its applications. The hydrogel uses a polypeptide-polymer copolymer as its backbone, and by adjusting the polymer network structure to match the mechanical properties of the nucleus pulposus, it achieves synergistic optimization of antioxidant properties, mechanical support, and biocompatibility. Because it does not rely on any drug bioactive factors, it can also effectively solve the problems of burst release and over-release of loaded drugs, exhibiting excellent overall performance and possessing broad market application potential.

Description

Technical Field

[0001] This invention relates to the field of medical technology, specifically to an injectable antioxidant hydrogel adapted to the mechanical properties of the nucleus pulposus, its preparation method, and its application. Background Technology

[0002] Intervertebral disc degeneration (IDD) is one of the main causes of low back pain. Its primary pathological changes originate from increased catabolism of the extracellular matrix (ECM) in the nucleus pulposus, leading to decreased water content in the nucleus pulposus. This disrupts the disc's biomechanical characteristics and causes abnormal stress within the disc, exacerbating the degeneration. In this process, abnormal nucleus pulposus cell metabolism and changes in the microenvironment trigger a vicious cycle. Elevated levels of free radical reactive oxygen species (ROS) and reactive nitrogen species (RNS) are among the hallmarks of this deteriorating microenvironment.

[0003] To address the above problems, existing technologies mainly focus on delivering antioxidant active ingredients through biomaterials to remove ROS and alleviate oxidative stress. However, these existing technologies still have obvious limitations: (1) Uncontrollable release behavior: Systems based on hydrogel-loaded active ingredients (nanozymes or drugs) generally exhibit initial burst release, leading to premature loss of antioxidant ingredients and an inability to maintain a long-term, stable, and effective concentration in the degenerated area, resulting in short-lived antioxidant effects; (2) Questionable long-term biocompatibility: Nanozymes and other strategies often involve metal-based nanomaterials, and their long-term retention in the body may raise safety concerns; (3) Limited mechanical properties and functions: Existing systems mostly focus on antioxidant functions and lack matching and support for the mechanical properties of degenerated nucleus pulposus tissue, making it difficult to restore the normal biomechanical function of the intervertebral disc; (4) Difficulty in achieving both injectability and stability: Many hydrogel materials, while striving to achieve good injectability, often sacrifice mechanical strength and in vivo stability, making it impossible to reside and function in the complex intervertebral disc microenvironment for a long time.

[0004] Therefore, there is an urgent need to develop a new type of biomaterial and its construction method that can achieve multiple synergistic effects, such as long-term removal of ROS and RNS, improvement of the degenerative microenvironment, good biocompatibility, injectability, and in vivo stability, while providing mechanical properties that match the nucleus pulposus tissue and providing appropriate mechanical support. This will promote the treatment of intervertebral disc degeneration towards a more comprehensive, effective, and stable direction. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a hydrogel. The hydrogel is an injectable hydrogel with a polypeptide-polymer copolymer as its backbone, which does not have the problem of burst release of loaded drugs; at the same time, by matching the mechanical properties of the nucleus pulposus with the tunable polymer network structure, it achieves synergistic optimization of antioxidant properties, mechanical support, and biocompatibility, resulting in superior overall performance and broad market application prospects.

[0006] This invention provides a polypeptide with the structure X1-Y(EYKY)2-X2.

[0007] X1 and X2 are independently selected from at least one of mercapto, azide, alkynyl, cycloalkynyl, tetrazinyl, transcyclooctenyl, norbornenyl, maleimide, vinyl sulfone, or an amino acid residue containing any of the above functional groups.

[0008] Among them, the core functional sequence unit "Y(EYKY)2" is relative to other units (e.g., F(EFKF)). n Or (GAGA) n Through its regularly arranged tyrosine residues, it exhibits stronger antioxidant activity; while the residues located at both ends... and This is used for covalent bonding with the polymer backbone. This design allows the resulting copolymer network to integrate the antioxidant function of the peptide with the structural characteristics of the polymer, thus achieving both controllable mechanical properties and good biocompatibility.

[0009] In some embodiments, the peptide has the structure CY(EYKY)2-C. Its tyrosine residues provide antioxidant activity, helping to protect the gel network and encapsulated cells from oxidative damage; the terminal cysteine ​​residues enable covalent cross-linking, constructing a robust mechanical support framework. The combination of these two elements results in a hydrogel that not only exhibits significant bioactivity but also possesses excellent mechanical stability and structural integrity.

[0010] The present invention also provides a method for preparing the polypeptide as described above, comprising:

[0011] The polypeptide is obtained by coupling individual amino acids or coupling segments according to the amino acid sequence of the polypeptide as described above;

[0012] The synthesis includes solid-phase synthesis, liquid-phase synthesis, or a combination of solid-phase and liquid-phase synthesis, and the present invention does not limit the specific methods used.

[0013] In some embodiments, the polypeptide is prepared using a solid-phase synthesis method.

[0014] In some embodiments, the preparation method further includes the steps of cutting the resin and purifying it.

[0015] The present invention also provides copolymers whose raw materials include the polypeptides as described above and at least one polymer;

[0016] The polymer comprises a polymer backbone and modifying groups;

[0017] The polymer backbone includes at least one of polyethylene glycol, polyvinyl alcohol, polylactic acid-glycolic acid copolymer or polycaprolactone copolymer;

[0018] The modifying group includes at least one of thiol, azide, alkynyl, cycloalkynyl, tetrazinyl, transcyclooctenyl, norbornyl, maleimide, or vinyl sulfone.

[0019] The copolymer possesses multiple synergistic advantages: firstly, its antioxidant properties stem from a stable, covalently linked polypeptide sequence, exhibiting long-lasting activity without the risk of burst release; secondly, its polymer network structure can be flexibly adjusted according to application to match the mechanical properties of target tissues such as the nucleus pulposus, providing physical support; and thirdly, the selected material components exhibit good biocompatibility. This design achieves synergistic optimization of antioxidant activity, controllable mechanical properties, and good biocompatibility.

[0020] In some embodiments, the polymer is two-arm maleimide polyethylene glycol, with a polymer backbone of polyethylene glycol and maleimide groups as the modifying groups.

[0021] The present invention also provides a method for preparing the copolymer as described above, which is obtained by reacting the polypeptide as described above with at least one of the polymers described above.

[0022] In some embodiments, the preparation method further includes adding a catalyst during the reaction. The catalyst includes tris(2-carboxyethyl)phosphine (TCEP) or Cu. + The addition of the catalyst can effectively promote the reaction and improve the yield and structural regularity of the copolymer.

[0023] In some embodiments, the reaction conditions include a shaking rate of 60–80 rad / min and a reaction time of 12–36 h. For example, the shaking rate is 60, 65, 70, 75, or 80 rad / min, and the reaction time is 12, 24, or 36 h. This shaking rate is optimized to ensure thorough and gentle mixing of the reaction system (peptide and polymer solution) compared to excessively high or low shaking rates. This reaction time is optimized to avoid problems such as incomplete reaction, decreased product performance, or increased energy consumption caused by excessively short or long reaction times.

[0024] In some specific embodiments, the oscillation speed of the reaction is 80 rad / min, and the reaction time is 24 h. This speed effectively promotes the contact and diffusion between reactant molecules, significantly improving reaction efficiency, while avoiding peptide chain breakage or polymer structure damage that may be caused by excessive shear force (e.g., excessively high rotation speed), or incomplete local reactions and product heterogeneity caused by uneven mixing (e.g., excessively low rotation speed). This reaction time avoids incomplete reactions and unstable product performance caused by too short a time, and also prevents side reactions or energy waste that may be caused by too long a time, thus ensuring both reaction quality and preparation efficiency.

[0025] In some embodiments, the preparation method further includes a dialysis purification step.

[0026] In some embodiments, the molar ratio of the peptide to the polymer is 1:1. This ratio is optimized to achieve an equivalence of reactants, which not only makes the cross-linking reaction more complete and avoids waste of raw materials, but also helps to form a network of cross-linking points with suitable density and uniform distribution, thereby obtaining a stable gel with regular structure and excellent mechanical properties.

[0027] The present invention also provides the use of the polypeptides, copolymers, or copolymers obtained by the preparation methods described above in the preparation of hydrogels.

[0028] Furthermore, the present invention also provides a hydrogel comprising medically acceptable excipients and at least one of the aforementioned polypeptides, copolymers, or copolymers obtained by the aforementioned preparation methods.

[0029] In some embodiments, the excipients include an acidic aqueous solution; the acidic aqueous solution is selected from at least one of hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, acetic acid, or citric acid. This acidic aqueous solution has been screened and optimized; compared to other acidic aqueous solutions, its mild acidic environment can effectively protect the structural integrity and antioxidant activity of the peptide, avoiding degradation and inactivation caused by strong acid conditions. Furthermore, the selected solution is stable, which is beneficial for the reproducibility of the process and the consistency of product quality in large-scale production.

[0030] In some embodiments, the content of the polypeptide, copolymer, or copolymer obtained by the preparation method described above is not less than 9% in the hydrogel. This content range has been experimentally verified. When the content is less than 9%, the crosslinking density is insufficient, and the system cannot form a stable three-dimensional network structure, i.e., it cannot successfully gel.

[0031] In some specific embodiments, the content of the aforementioned polypeptide, copolymer, or copolymer obtained by the aforementioned preparation method in the hydrogel is 10%. The hydrogel formed at this concentration has rheological properties (such as storage modulus G') that are highly matched with natural human nucleus pulposus tissue, achieving mechanical biocompatibility between the implant material and the host tissue, and providing suitable mechanical support for intervertebral disc repair.

[0032] In other specific embodiments, the content of the aforementioned peptide, copolymer, or copolymer obtained by the aforementioned preparation method in the hydrogel is 20%. At this concentration, the crosslinking reaction is significantly accelerated, the gelation time can be shortened to less than 1 minute, and the storage modulus exceeds the storage modulus of the nucleus pulposus by an order of magnitude, providing a reliable material basis for applications requiring higher mechanical strength (such as repair of severe defects or high-load sites).

[0033] Furthermore, the present invention also provides a method for preparing the hydrogel as described above, comprising:

[0034] The copolymer as described above, or the copolymer obtained by the preparation method described above, is dissolved in the acidic aqueous solution, and the pH value is adjusted to 6.0~9.0 to obtain the hydrogel.

[0035] In some embodiments, the pH value is adjusted to 6.0–9.0 to obtain the hydrogel. For example, the pH value is adjusted to 6.0, 7.0, 8.0, or 9.0. Under these conditions, the solution can gel within 10 minutes to form a structurally stable hydrogel.

[0036] Preferably, the pH value is adjusted to 7.0~7.5, and the gelation environment is controlled in the near-neutral to weakly alkaline range. On the one hand, this avoids the destruction of the antioxidant activity of peptides by strong acid / strong alkali conditions, and on the other hand, it ensures that the gel product has excellent biocompatibility and will not cause additional acid or alkali stimulation to the surrounding tissues.

[0037] In some embodiments, the preparation method further includes an ultrasonic step to promote the dissolution of the copolymer in an acidic aqueous solution and ensure uniform mixing of the components.

[0038] Furthermore, the present invention provides the use of the polypeptide, copolymer, copolymer obtained by the preparation method described above, hydrogel, or hydrogel obtained by the preparation method described above in the preparation of medical products.

[0039] In some embodiments, the medical device functions to include at least one of the following:

[0040] For the repair, filling or support of flexible tissues, including intervertebral discs, cartilage, menisci or soft tissue of the brain;

[0041] Used to prevent and / or treat oxidative stress damage to tissues;

[0042] Provides physiologically appropriate mechanical support to damaged or degenerated tissues;

[0043] Used to treat and / or improve intervertebral disc degeneration and related diseases.

[0044] Furthermore, the present invention also provides medical products, including the polypeptides as described above, the copolymers as described above, the copolymers obtained by the preparation methods as described above, the hydrogels as described above, or the hydrogels obtained by the preparation methods as described above, and other therapeutic agents.

[0045] In some embodiments, the therapeutic agent includes at least one of the following:

[0046] Anti-inflammatory and analgesic drugs used to relieve local inflammation and pain, such as nonsteroidal anti-inflammatory drugs (e.g., ketoprofen, diclofenac sodium), glucocorticoids (e.g., dexamethasone), or local anesthetics (e.g., lidocaine).

[0047] Nutritional and metabolic drugs used to improve the cellular metabolic environment of degenerated tissues, such as glucose, amino acids, chondroitin sulfate, or hyaluronic acid.

[0048] Tissue regeneration promoters used to stimulate cell proliferation and extracellular matrix synthesis, such as growth factors (e.g., TGF-β3, BMP-2, bFGF), cytokines, or bioactive peptides with chondroprotective effects.

[0049] The hydrogel can be used alone as an excellent mechanical support and antioxidant carrier, and can also serve as a functional delivery platform to achieve physical encapsulation or chemical combination with the aforementioned therapeutic agents, thereby constructing a combined product with multiple functions of "mechanical repair, oxidative stress intervention and drug sustained release", providing a synergistic treatment solution for complex pathological processes such as intervertebral disc degeneration.

[0050] Furthermore, the present invention also provides a method for treating and / or improving intervertebral disc degeneration and related diseases, the method comprising administering an effective dose of the hydrogel or medical product as described above to a subject in need.

[0051] In some embodiments, the administration is performed via local injection.

[0052] In some embodiments, the object of the offering includes humans or mammals. The mammals include, but are not limited to, orangutans, gibbons, baboons, macaques, marmosets, mice, rats, guinea pigs, hamsters, rabbits, dogs, cats, pigs, sheep, horses, cattle, and camels.

[0053] This invention relates to an injectable antioxidant hydrogel adapted to the mechanical properties of the nucleus pulposus, its preparation method, and its applications. This hydrogel enables controlled release of the active ingredient, avoiding the burst release and over-release problems of drugs in traditional loading systems. Simultaneously, its polymer network structure is tunable, capable of matching the mechanical properties of the nucleus pulposus tissue, thereby synergistically optimizing antioxidant function, mechanical support, and biocompatibility. This hydrogel exhibits outstanding comprehensive performance and has promising application prospects. Attached Figure Description

[0054] Figure 1 The structure of the CY(EYKY)2C polypeptide;

[0055] Figure 2 Mass spectrometry results for CY(EYKY)2C peptide;

[0056] Figure 3 For CY(EYKY)2C peptide 1 H-NMR results;

[0057] Figure 4 FT-IR results for the CY(EYKY)2C peptide;

[0058] Figure 5 The images show the FT-IR spectra of reactants and products before and after the click chemical polymerization reaction (Figure A is the full FT-IR spectrum, and Figures B and C are magnified views of the FT-IR spectra).

[0059] Figure 6 This refers to the hydrogel formation process;

[0060] Figure 7 The results show the rheological properties of the hydrogel;

[0061] Figure 8 Results for the injectability properties of the hydrogel;

[0062] Figure 9 For Y (EYKY) n -Solubility of PEG in water under different conditions, at a mass concentration of 1%;

[0063] Figure 10 Y (EYKY) under pH changes n -Solubility of PEG at a mass concentration of 1%;

[0064] Figure 11 The result is the lowest gel test concentration of Y(EYKY)2-PEG;

[0065] Figure 12 The gelation time and rheological properties of Y(EYKY)2-PEG hydrogels with different concentrations were tested.

[0066] Figure 13 The results show the antioxidant properties of the hydrogel;

[0067] Figure 14 This demonstrates the effect of hydrogels in preventing intracellular ROS accumulation.

[0068] Figure 15 For the results of animal experiments, (A) 4 weeks, (B) 8 weeks, (C) MRI score, (D) intervertebral disc height index (DHI).

[0069] Figure 16 The results show the anti-swelling properties of Y(EYKY)2-PEG hydrogel. Detailed Implementation

[0070] This invention provides an injectable antioxidant hydrogel adapted to the mechanical properties of the nucleus pulposus, its preparation method, and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0071] Unless otherwise defined in this invention, scientific and technical terms related to this invention shall have the meanings understood by one of ordinary skill in the art.

[0072] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0073] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0074] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0075] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0076] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0077] In this invention, the effective dose includes a "therapeutic effective dose" or a "preventive effective dose," which refers to an amount sufficient, within reasonable medical judgment, to treat and / or improve intervertebral disc degeneration and related diseases while avoiding serious side effects sufficiently (within a reasonable benefit / risk ratio). The preventive or therapeutic effective dose of a drug will vary depending on factors such as the specific drug selected (e.g., considering the drug's potency, effectiveness, and half-life), the chosen route of administration, the disease being prevented or treated, the severity of the disease being prevented or treated, the age, size, weight, and physical condition of the patient being prevented or treated, the patient's medical history, the duration of prevention or treatment, the nature of concurrent therapies, and the desired preventive or therapeutic effect, but can still be routinely determined by those skilled in the art.

[0078] This invention addresses the problems in current treatment of intervertebral disc degeneration, such as insufficient antioxidant properties of basic biomaterials, mismatch between mechanical properties and nucleus pulposus tissue, and difficulty in balancing biocompatibility and injectability, and proposes a new solution.

[0079] This invention constructs an injectable hydrogel with a polypeptide-polymer copolymer backbone. The antioxidant properties are derived from the phenolic hydroxyl groups on the side chains of the amino acid residues of the polypeptide itself, which are inherent properties of the backbone and do not have the problem of burst release of loaded drugs. At the same time, by matching the mechanical properties of the nucleus pulposus with the tunable polymer network structure, the synergistic optimization of antioxidant properties, mechanical support and biocompatibility is achieved.

[0080] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0081] The present invention will be further illustrated below with reference to the embodiments:

[0082] Example 1: Synthesis and Identification of Y(EYKY)2-PEG Copolymer

[0083] 1.1 Synthesis of CY(EYKY)2C peptide

[0084] The peptides were synthesized using a CEM Liberty Blue fully automated continuous microwave peptide analyzer (USA) at a concentration of 0.20 M. The amounts of various amino acids were calculated based on the preset sequence, and the corresponding protected amino acids were weighed and dissolved in a certain amount of DMF. The peptides were dissolved by sonication. After dissolution, a reagent tube was attached to the instrument. Separately, 14.21 g of Oxime solid was weighed and dissolved in 100 mL of DMF to prepare an Oxime solution. 7.8 mL of DIC solution was mixed with 92.2 mL of DMF to prepare a DIC solution. Oxime and DIC were used as coupling agents. Finally, 90 mL of 20% piperidine solution was measured as a deprotection solvent. Finally, 0.596 mg of Rink amide AM resin (degree of substitution: 0.338) was weighed and placed in the reaction vessel. (CEM Liberty Blue) The Blue fully automated continuous microwave peptide analyzer performs the following steps for peptide synthesis: swell the resin, add the deprotecting solvent, turn on microwave heating (to 90°C), remove the deprotecting solvent, wash three times, add the corresponding amino acid, add the coupling agent, turn on microwave heating (to 90°C, react for 110 or 230 seconds), and wash three times with DMF. This constitutes one amino acid coupling process. There are a total of 16 amino acids in the sequence. This process is repeated 16 times to complete the solid-phase synthesis.

[0085] Resin cleavage: After solid-phase synthesis, 10 mL of lysis buffer containing 9.25 mL trifluoroacetic acid, 0.25 mL water, 0.25 mL 1,2-ethylenedithiol, and 0.25 mL triisopropylsilane was added to the resin. The mixture was then placed on a rotary osmometer for lysis for 2 hours. After lysis, 30 mL of anhydrous diethyl ether was added and thoroughly mixed. The mixture was then centrifuged at 8000 rpm for 5 minutes. This process was repeated three times to obtain a white crude polypeptide product. The cleavage site was the junction of the amino and dibenzyl groups on the dendrite.

[0086] Crude product purification: The crude product was dissolved in a 10 mg / mL mixed solvent, with a water-to-acetonitrile ratio of 6:4. Purification was then performed using preparative liquid chromatography (Shimadzu Preparative Liquid Chromatography, Japan). Mobile phase A consisted of acetonitrile, and mobile phase B consisted of water containing 0.1% TFA. The elution gradient was 80%–20% water, 20%–80% acetonitrile, for 30 minutes. The purified product was collected based on the absorption peak at approximately 20 minutes.

[0087] The obtained pure product solution was subjected to rotary evaporation to remove acetonitrile and most of the water, leaving 20 mL of water. The solution was then cooled in liquid nitrogen and subsequently placed in a freeze dryer at a cold trap temperature of -80°C and a vacuum of 1.0 Pa for 72 hours to obtain pure product powder.

[0088] The structure of the CY(EYKY)2C polypeptide is as follows: Figure 1 As shown.

[0089] 1.2 Identification of Polypeptides

[0090] 1. The purified lyophilized polypeptide was a white powder.

[0091] 2. The theoretical relative molecular mass of the CY(EYKY)2C polypeptide is 1554 Da.

[0092] 3. Maldi-TOF-MS analysis showed that the relative molecular mass of the peptide after hydrogenation was 1555.6 Da, which matches the theoretical value. Figure 2 ).

[0093] 4. 1 1H-NMR spectroscopy: The sample was dissolved in deuterated DMSO (d6) and measured at 400 MHz, with more than 256 measurements. The results are shown below. Figure 3 .

[0094] 5. The Fourier Transform Infrared (FT-IR) spectra of the CY(EYKY)2C peptide are as follows: Figure 4 As shown.

[0095] 1.3 Synthesis and Identification of Y(EYKY)2-PEG Copolymer

[0096] 1. The synthesized CY(EYKY)2C peptide was polymerized with two-arm maleimide polyethylene glycol (Mal-PEG-Mal, Mw=5kDa) via click chemistry to form a copolymer. The synthesis method is as follows:

[0097] Weigh out the reactants CY(EYKY)2C and Mal-PEG-Mal in a 1:1 molar ratio, dissolve them in deionized water, and add 0.2 equivalents of tris(2-carboxyethyl)phosphine (TCEP). React on a shaker (60-80 rad / min) for 24 hours. After the reaction is complete, remove the reaction solution and dialyze it against deionized water for 2-4 days to remove unreacted monomers. After dialysis, freeze-dry the solution to obtain the Y(EYKY)2-PEG copolymer.

[0098] 2. The synthesis of the Y(EYKY)2-PEG copolymer is characterized by the consumption of the conjugated structures of the CY(EYKY)2C peptide's biterminal thiol (-SH) groups and the Mal-PEG-Mal biterminal maleimide groups. Its FT-IR results are as follows: Figure 5 As shown.

[0099] FT-IR results showed that after the polymerization reaction, the 1710 cm⁻¹ of Mal-PEG-Mal... -1 The indicated maleimide structure disappears, and the ~2560 cm⁻¹ generated by the -SH vibration... -1 The disappearance of characteristic absorption indicates that the click chemical reaction is complete and the molecular chain polymerization is successful.

[0100] Example 2: Preparation and Rheological Properties of Injectable Hydrogels

[0101] 1. The preparation method of the injectable hydrogel is as follows: The synthesized Y(EYKY)2-PEG copolymer polymer is ultrasonically dissolved in an acidic aqueous solution at a concentration of 10 wt%. After complete dissolution, the pH is adjusted to alkaline, and the gel will form within 10 minutes (e.g., Figure 6 (As shown).

[0102] 2. The rheological properties of hydrogels, such as Figure 7 As shown, when the scanning speed is close to 1 Hz, its storage modulus G' is approximately 400 Pa, which matches the natural nucleus pulposus.

[0103] 3. This hydrogel can be easily dispensed using a syringe, exhibiting excellent injectability. Figure 8 ).

[0104] Example 3: Filtering the number of repetitions (n ​​value) of the core repeating unit EY KY

[0105] Y (EYKY) n - PEG with a repeating sequence of EYKY with 1 repeat can dissolve in water but cannot form a gel; with 3 repeats, it cannot dissolve under mild conditions, thus failing to meet the gelation prerequisite. The test results are as follows: Figure 9 and Figure 10 .

[0106] Example 4: Minimum gelation concentration test of Y(EYKY)2-PEG hydrogel

[0107] An experiment was conducted to test the lowest gelling concentration of Y(EYKY)2-PEG hydrogel. Results showed that gelation failed below 9% concentration, rheological properties matched those of the nucleus pulposus at 10% concentration, and gelation accelerated to within 1 minute at 20% concentration, with the storage modulus exceeding that of the nucleus pulposus by an order of magnitude. Data are as follows: Figure 11 and Figure 12 As shown.

[0108] Example 5: Antioxidant properties of hydrogels

[0109] 1. The antioxidant properties of the hydrogel were tested using DPPH free radical assays, and the results are as follows: Figure 13 As shown, the hydrogel can achieve a free radical scavenging rate of over 85% within 24 hours.

[0110] 2. Plate nucleus pulposus cells into 24-well plates (10... 5 Cells were co-cultured with the hydrogel after being enclosed in wells (each cell / well) and simultaneously stimulated with 100 μM hydrogen peroxide (H2O2) to simulate the highly oxidative microenvironment within the nucleus pulposus of a degenerated intervertebral disc. Intracellular reactive oxygen species were tested using a DCFH-DA (2',7'-dichlorofluorescein diacetate) kit after 24 hours. The results are as follows: Figure 14As shown.

[0111] The results indicate that intracellular ROS accumulates (green fluorescence) under H2O2 stimulation, and co-culturing with Y(EYKY)2-PEG hydrogel can significantly reduce this phenomenon, suggesting that the hydrogel can prevent excessive ROS accumulation in nucleus pulposus cells in a strongly oxidizing environment.

[0112] Example 6: The effect of hydrogel on improving intervertebral disc degeneration (animal experiment)

[0113] Intervertebral disc degeneration was induced in SD rats by acupuncture at the caudal intervertebral disc location (disc location confirmed by palpation; a 22G needle was inserted directly into the nucleus pulposus to a depth of approximately 5 mm, held for 30 seconds, then rotated 180° and held for another 30 seconds). After modeling, 20 μL of hydrogel was injected for 4 and 8 weeks. The treatment effect was analyzed and data quantified using T2-weighted magnetic resonance imaging (MRI) and MicroCT. Results are as follows: Figure 15 As shown.

[0114] The results show that injecting this polypeptide hydrogel can effectively slow down the loss of water signals inside the intervertebral disc and help maintain the height of the intervertebral space.

[0115] Example 7: Anti-swelling properties of hydrogel

[0116] The hydrogel's anti-swelling properties are as follows: Figure 16 As shown, the results indicate that the internal network of this material is stable and can be maintained in water.

[0117] Based on the above technical solution, the advantages of the present invention are as follows:

[0118] 1. Stable antioxidant properties: Antioxidant groups are embedded in the backbone rather than physically incorporated, avoiding loss;

[0119] 2. Matching mechanical properties: By adjusting the hydrogel concentration ratio and gelation process, the storage modulus is made close to that of the nucleus pulposus tissue;

[0120] 3. The entire process is chemically synthesized without the use of any biological machinery, which can significantly reduce purification complexity and enhance batch-to-batch stability.

[0121] This invention provides an injectable antioxidant hydrogel adapted to the mechanical properties of the nucleus pulposus, its preparation method, and its applications. The hydrogel uses a polypeptide-polymer copolymer as its backbone, which not only effectively solves the problems of burst release and over-release of loaded drugs, but also allows for the adjustment of the polymer network structure to match the mechanical properties of the nucleus pulposus, achieving synergistic optimization of antioxidant properties, mechanical support, and biocompatibility. It exhibits excellent overall performance and has broad market application potential.

[0122] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide, characterized in that, The structure of the polypeptide is CY(EYKY)2-C.

2. A copolymer, characterized in that, The polypeptide of claim 1 and the two-arm maleimide polyethylene glycol are reacted to obtain the polypeptide and the two-arm maleimide polyethylene glycol. The molar ratio of the polypeptide to the two-arm maleimide polyethylene glycol is 1:

1.

3. The method for preparing the copolymer according to claim 1, characterized in that, The polypeptide of claim 1 is prepared by reacting it with two-arm maleimide polyethylene glycol; the molar ratio of the polypeptide to the polymer is 1:

1.

4. The preparation method according to claim 3, characterized in that, The reaction conditions include: a oscillation rate of 60-80 rad / min and a reaction time of 12-36 h.

5. The use of the copolymer of claim 2 in the preparation of hydrogels.

6. A hydrogel, characterized in that, This includes excipients acceptable in the field of medical technology and copolymers as described in claim 2.

7. The hydrogel according to claim 6, characterized in that, The excipients include an acidic aqueous solution; the acidic aqueous solution is selected from at least one of hydrochloric acid, phosphoric acid, sulfuric acid, oxalic acid, acetic acid or citric acid.

8. The hydrogel according to claim 6, characterized in that, The copolymer content of claim 2 is not less than 9%.

9. A method for preparing the hydrogel according to any one of claims 6 to 8, characterized in that, include: The copolymer described in claim 2 is dissolved in an acidic aqueous solution, and the pH value is adjusted to 6.0~9.0 to obtain the hydrogel.

10. The hydrogel according to any one of claims 6 to 8, or the hydrogel obtained by the preparation method of claim 9, is used in the preparation of medical products; the medical products include at least one of the following functions: For the repair, filling or support of flexible tissues, including intervertebral discs, cartilage, menisci or soft tissue of the brain; Provides physiologically appropriate mechanical support to damaged or degenerated tissues; Used to treat and / or improve intervertebral disc degeneration and related diseases.

11. Medical supplies, characterized in that, Includes the hydrogel according to any one of claims 6 to 8, or the hydrogel obtained by the preparation method of claim 9.

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