Core-shell microneedle for treating intervertebral disc degeneration and preparation method thereof

Abstract

The invention discloses a core-shell microneedle for treating intervertebral disc degeneration and a preparation method of the core-shell microneedle, and relates to the technical field of biological medicines. The core-shell microneedle comprises a substrate and a plurality of microneedles arranged on the substrate, each microneedle has a core-shell structure, the core-shell structure comprises a shell layer and a core layer, the shell layer comprises a metal phenolic aldehyde self-driven nano-motor and adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid, and the core layer comprises a metal phenolic aldehyde self-driven nano-motor and adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid. The metal phenolic aldehyde self-driven nano motor is a metal phenolic aldehyde complex network TA (at) MgO2 composed of tannic acid and magnesium peroxide, the chemical formula of the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid is shown in the specification, and the TA (at) MgO2 is dispersed in the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid; the core layer comprises a porous slow-release hydrogel, and the porous slow-release hydrogel is loaded with a drug quercetin through the host-guest interaction. The core-shell microneedle provided by the invention can reduce the intracellular ROS level of degenerative fibrous ring cells and recover the activity of AF cells.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a core-shell microneedle for treating intervertebral disc degeneration and a preparation method thereof. BACKGROUND

[0002] Intervertebral disc degeneration (IDD) is one of the main causes of low back pain. The annulus fibrosus, as the outer mechanical barrier of the intervertebral disc, is crucial for maintaining disc height, distributing load and preventing nucleus pulposus herniation. Traditional surgical treatments such as suturing or artificial material implantation can only temporarily seal the defect and cannot restore the layered structure and biomechanical function of the annulus fibrosus, and there is a risk of large trauma, high recurrence rate and accelerated adjacent segment degeneration.

[0003] Studies have shown that ferroptosis is an important factor leading to the reduction of annulus fibrosus cells, local inflammatory response and imbalance of extracellular matrix metabolism, and inhibiting ferroptosis can reduce intracellular iron content and lipid peroxidation level, thereby promoting annulus fibrosus repair. Therefore, maintaining the mechanical properties of the annulus fibrosus, controlling the inflammatory response and inhibiting ferroptosis may be a potential approach for annulus fibrosus repair. It is currently believed that pathological microenvironments (such as oxidative stress, inflammatory factor infiltration and iron overload) can exacerbate IDD progression by inducing cell ferroptosis, and targeting the regulation of ferroptosis provides a new approach for annulus fibrosus repair. Ferroptosis is an iron-dependent form of programmed cell death characterized by accumulation of lipid peroxidation and imbalance of antioxidant defense system. Numerous studies have confirmed that excessive generation of reactive oxygen species (ROS) and iron metabolism disorders in the local microenvironment can activate the key pathways of ferroptosis. In addition, ferroptosis induced by pathological microenvironment not only affects cell survival, but also leads to upregulation of matrix-degrading enzyme expression, damaging the structure and function of the annulus fibrosus.

[0004] However, traditional drug delivery systems are difficult to penetrate the dense structure of the annulus fibrosus, resulting in insufficient local concentration of drugs; and the biological materials such as exosomes and nanoparticles loaded by traditional microneedles mainly rely on free diffusion for treatment, which has the problem of low bioavailability and delivery efficiency.

[0005] Microneedle technology as an innovative platform for transdermal drug delivery has made significant progress in the field of annulus fibrosus repair in recent years. However, there are still the following limitations in the use of microneedles in tissue repair, especially in the treatment of intervertebral disc degeneration at the present stage:

[0006] 1. Mechanical mismatch and insufficient tissue integration, current single-layer microneedles mostly adopt homogeneous straight needle design, which does not match the mechanical properties of the annulus fibrosus (collagen fibers arranged at 30°) and is prone to displacement or shedding after implantation. Moreover, the peel adhesion of traditional smooth microneedles in a dynamic mechanical environment (such as spinal flexion and extension activities) is only 0.29 N, while the threaded microneedle is improved to 0.47 N, but still does not reach the safety threshold of the high mechanical stress environment (>1 MPa) of the annulus fibrosus.

[0007] 2. Limited delivery control and synergistic therapy, existing technologies rely on passive diffusion (such as laminin adsorption) or external stimulation (such as near-infrared light, manual pressure), and cannot respond to microenvironment pathological changes (such as pH decrease, inflammation factor increase) for intelligent drug release. At the same time, single therapy (such as only delivering exosomes or anti-inflammatory drugs) is difficult to cope with the multifactorial pathological process of annulus fibrosus degeneration (including mitochondrial dysfunction, inflammatory cascade, and matrix metabolism imbalance).

[0008] 3. Insufficient maintenance of biological activity, the hypoxic, low-pH, and high-active oxygen microenvironment of the degenerative annulus fibrosus can significantly reduce the effectiveness of functional drugs, and existing ordinary single-layer microneedles lack targeted protection strategies.

[0009] 4. Insufficient penetration strength of deep tissue, the annulus fibrosus surface is dense, and the ordinary single-layer microneedle compression modulus is only 50-100 kPa, making it difficult to effectively penetrate.

[0010] 5. Short residence time, ordinary single-layer microneedles are prone to premature degradation under body fluid flushing and dynamic load, making it difficult to cover the annulus fibrosus repair period.

[0011] In recent years, the integration of biomaterials and regenerative medicine has provided new ideas for annulus fibrosus repair, but most strategies are difficult to achieve the multiple goals of "precise delivery-mechanical support-long-term regeneration". SUMMARY

[0012] To solve the technical problems existing in the prior art, the embodiments of the present application provide a core-shell microneedle for treating intervertebral disc degeneration and a preparation method thereof. The technical solution is as follows:

[0013] A core-shell microneedle for treating intervertebral disc degeneration, the core-shell microneedle comprises a substrate and a plurality of microneedles arranged on the substrate, each microneedle has a core-shell structure, the core-shell structure comprises a shell layer and a core layer, the shell layer comprises a metal phenolic self-driven nanomotor and adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid, the metal phenolic self-driven nanomotor is a metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide, and the chemical formula of the adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid is as follows:

[0014] ,

[0015] wherein n is 500-2500, the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide is dispersed in the adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid; and

[0016] The core layer comprises a porous sustained-release hydrogel loaded with a drug quercetin through host-guest interaction.

[0017] Optionally, the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide is formed by the following method: Mg 2+ The phenolic hydroxyl groups of tannic acid are covered on the surface of MgO2 particles through coordination to form a dense cross-linked metal phenolic network, thereby obtaining the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide; or the preparation method of the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide is as follows: mixing a tannic acid solution with a suspension of magnesium peroxide, and complexing through ultrasonic assistance to obtain the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide.

[0018] Optionally, the adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid is prepared by the following method: adding dihydrocaffeic acid HCA to the adipic dihydrazide modified hyaluronic acid HA-ADH solution, and reacting to obtain the adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid HA-ADH-HCA, whose chemical formula is as follows:

[0019] ;

[0020] And / or, the mass ratio of the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide to the adipic dihydrazide-dihydrocaffeic acid modified hyaluronic acid is 1:5-1:10, or 1:10.

[0021] Optionally, the porous sustained-release hydrogel is β-cyclodextrin modified methacrylated gelatin GelMA-β-CD; and / or, the β-cyclodextrin modified methacrylated gelatin GelMA-β-CD is prepared by the following method:

[0022] a) adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to the β-cyclodextrin solution, and reacting to obtain carboxyl-activated β-cyclodextrin;

[0023] b) adding the carboxyl-activated beta-cyclodextrin to the methacrylated gelatin solution, reacting, thereby obtaining the beta-cyclodextrin-modified methacrylated gelatin GelMA-beta-CD, whose chemical reaction formula is shown as follows:

[0024] ;

[0025] And / or, the beta-cyclodextrin-modified methacrylated gelatin GelMA-beta-CD forms an inclusion compound with quercetin through host-guest interaction, wherein the mass ratio of the quercetin to the beta-cyclodextrin-modified methacrylated gelatin GelMA-beta-CD is 1:1-1:5, or 1:2.

[0026] The preparation method of the core-shell microneedle, the method comprising:

[0027] (1) preparing a metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide: mixing a tannic acid solution with a suspension of magnesium peroxide, and allowing a complexation reaction to occur through ultrasonic assistance, thereby obtaining the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide;

[0028] (2) synthesizing a shell layer material TA@MgO2@HA: adding the TA@MgO2 obtained in step (1) to a hexanedioic dihydrazide-dihydrocaffeic acid-modified hyaluronic acid solution, and using ultrasonic treatment to mix the solution, so that the TA@MgO2 is uniformly dispersed, thereby obtaining the shell layer material TA@MgO2@HA;

[0029] (3) synthesizing a core layer material: the core layer material comprises a porous sustained-release hydrogel, and the porous sustained-release hydrogel loads the drug quercetin through host-guest interaction;

[0030] (4) assembling into a core-shell microneedle: injecting the shell layer material TA@MgO2@HA into a mold, adding a solution of the core layer material after drying, and then demolding after drying, thereby obtaining the core-shell microneedle.

[0031] Optionally, in step (1), the molar ratio of the tannic acid to the magnesium peroxide is 1:1-1:5, or 1:3; and / or the power of the ultrasonic is 200 W-400 W, or 200 W; and / or the time of the ultrasonic is 20-40 min, or 40 min.

[0032] Optionally, in step (2), the mass ratio of the TA@MgO2 to the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid is 1:5-1:10, or 1:10; and / or, the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid is prepared by adding dihydrocaffeic acid HCA to the adipic acid dihydrazide modified hyaluronic acid HA-ADH solution, reacting, thereby obtaining the hydrazide-dihydrocaffeic acid double-modified hyaluronic acid HA-ADH-HCA, the chemical formula of which is as follows:

[0033] .

[0034] Optionally, in step (3), the porous sustained-release hydrogel is β-cyclodextrin modified methacrylated gelatin GelMA-β-CD, and / or the β-cyclodextrin modified methacrylated gelatin GelMA-β-CD is prepared by the following method:

[0035] a) adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide to the β-cyclodextrin solution, reacting, thereby obtaining carboxyl-activated β-cyclodextrin;

[0036] b) adding the carboxyl-activated β-cyclodextrin to the methacrylated gelatin solution, reacting, thereby obtaining the β-cyclodextrin modified methacrylated gelatin GelMA-β-CD, the chemical formula of which is as follows:

[0037] ;

[0038] and / or, mixing the quercetin anhydrous ethanol solution with the β-cyclodextrin modified methacrylated gelatin GelMA-β-CD solution to form a clathrate solution, thereby obtaining the core layer material QUE@GelMA;

[0039] and / or, the mass ratio of the quercetin to the β-cyclodextrin modified methacrylated gelatin GelMA-β-CD is 1:1-1:5, or 1:2.

[0040] An adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid, the chemical formula of which is as follows:

[0041] ,

[0042] wherein n is 500-2500.

[0043] The method for preparing adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid includes: adding dihydrocaffeic acid (HCA) to a solution of the adipic acid dihydrazide-modified hyaluronic acid (HA-ADH), reacting to obtain the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid (HA-ADH-HCA), the chemical formula of which is shown below:

[0044] .

[0045] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0046] The beneficial effects of preparing self-driven nanoparticles TA@MgO2: Self-driven gas-generating nanoparticles TA@MgO2 with a shell composed of a metal-phenolic network can thrive in the fibrous ring AF microenvironment. + It reacts with MgO2 to release O2, thereby triggering a self-driven gas production effect in an acidic microenvironment. It penetrates deep into the damaged area to release oxygen, improve hypoxia, and improve the acidic microenvironment of degenerated AF. At the same time, the present invention confirms through DPPH free radical scavenging experiment, SOD enzyme activity detection experiment and H2O2 scavenging experiment that the antioxidant properties of TA can effectively scavenge ROS and inhibit Fenton reaction.

[0047] Beneficial Effects of Assembled Core-Shell Microneedles TMH / QG-MN: Nucleotomy is commonly used in the clinical treatment of degenerated intervertebral discs (IDDs), but the lack of treatment for damaged aneurysms (AFs) leads to continuous leakage of the nucleus pulposus, requiring secondary surgery. Currently, biomaterials for treating the annulus fibrosus lack temporal and responsiveness. This invention employs a core-shell domain loading strategy to prepare core-shell microneedles. The outer shell is a high-hardness material to ensure puncture capability; the core is a porous, sustained-release hydrogel that can load therapeutic components, achieving a match with the mechanical properties of the annulus fibrosus. Leveraging its temporal degradation characteristics, it sequentially reacts with TA@MgO2 in the acidic microenvironment of the degenerated annulus fibrosus, generating asymmetric thrust to drive motor movement, improving the hypoxic environment, reducing ROS levels, and improving the extracellular matrix microenvironment of the annulus fibrosus. Then, it restores AF cell viability by releasing core-layer functional drugs. During IDD treatment, the core-shell microneedles not only act as a sealant to prevent further leakage of the nucleus pulposus but also spontaneously improve the microenvironment of the degenerated annulus fibrosus and restore cell function, thereby promoting intervertebral disc repair. In addition, the core-shell microneedles TMH / QG MN of the present invention can reduce the intracellular ROS level of degenerated annulus fibrosus cells through synergistic effects, thus avoiding cellular oxidative stress. Attached Figure Description

[0048] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without any creative effort on the basis of these drawings.

[0049] Figures 1A-1F is the apparent morphology feature and basic physical and chemical property diagram of the self-driven nanomotor TA@MgO2 provided by the embodiment 1 of the present application; wherein, Figure 1A is the transmission electron microscope diagram characterizing the surface morphology of the metal phenolic self-driven nanomotor TA@MgO2; Figure 1B is the X-ray diffraction (XRD) diagram analyzing the physical structure of MgO2 and TA@MgO2; Figure 1C is the Zeta potential diagram of TA, MgO2 and TA@MgO2, further embodying the surface charge characteristic of TA@MgO2; Figures 1D-1F is the X-ray photoelectron spectroscopy (XPS) diagram further confirming the chemical composition of TA@MgO2;

[0050] Figures 2A-2D is the oxidation resistance characteristic diagram of the self-driven nanomotor TA@MgO2 provided by the embodiment 2 of the present application; wherein, Figure 2A is the DPPH free radical scavenging experiment result diagram of the self-driven nanomotor TA@MgO2; Figure 2B is the diagram of the DPPH scavenging ability of the self-driven nanomotor TA@MgO2; Figure 2C is the SOD-like activity diagram of TA@MgO2; Figure 2D is the H2O2 scavenging ability diagram of TA@MgO2;

[0051] Figures 3A-3E is the synthesis characterization diagram of the core-shell microneedle precursor solution provided by the embodiment 3 of the present application; wherein, Figure 3A is the chemical synthesis step diagram of hydrazide (ADH)-dihydrocaffeic acid (HCA) modified hyaluronic acid (HA-ADH-HCA); Figure 3B is the chemical synthesis step diagram of β-cyclodextrin (β-CD) modified methacrylated gelatin (GelMA-β-CD); Figure 3C is the nuclear magnetic resonance hydrogen spectrum diagram of hydrazide (ADH) modified HA; Figure 3D is the nuclear magnetic resonance hydrogen spectrum diagram of hydrazide (ADH)-dihydrocaffeic acid (HCA) modified HA (HA-ADH-HCA); Figure 3E is the nuclear magnetic resonance hydrogen spectrum diagram of β-cyclodextrin (β-CD) modified GelMA (GelMA-β-CD);

[0052] Figures 4A-4Cis the fluorescence microscope image and degradation behavior chart of the core layer, shell layer and core-shell microneedle provided in Embodiment 4 of the present application; wherein, Figure 4A is the fluorescence microscope image of the core layer microneedle QUE@GelMA MN (green fluorescence), the shell layer microneedle TA@MgO2@HA MN (red fluorescence) and the core-shell microneedle TMH / QG MN; Figure 4B is the chart of hematoxylin & eosin (H&E) staining of the core-shell microneedle TMH / QG MN punctured into the pig skin section; Figure 4C is the chart of the degradation behavior of the core-shell microneedle TMH / QG MN;

[0053] Figures 5A-5B is the chart of the functional drug molecule release of the core-shell microneedle TMH / QG MN provided in Embodiment 5 of the present application; wherein, Figure 5A is the chart of the tannic acid (TA) release of the core-shell microneedle TMH / QG MN placed in a normal physiological microenvironment (pH 7.4), an acidic microenvironment (pH 6.5), a rich active oxygen (ROS) microenvironment and a degenerative annulus fibrosus microenvironment (pH 6.5+ROS) for 14 days; Figure 5B is the chart of the quercetin (QUE) release of the core-shell microneedle TMH / QG MN placed in a normal physiological microenvironment (pH 7.4), an acidic microenvironment (pH 6.5), a rich active oxygen (ROS) microenvironment and a degenerative annulus fibrosus microenvironment (pH 6.5+ROS) for 14 days;

[0054] Figure 6 is the chart of the intracellular active oxygen level test of the annulus fibrosus cells co-cultured with the core layer, shell layer and core-shell microneedle for 48 hours under oxidative stress induced, provided in Embodiment 6 of the present application;

[0055] Figure 7A is the chart of the X-ray and MRI imaging data of the rat tail vertebrae at 4 weeks and 8 weeks after the operation, provided in Embodiment 7 of the present application; Figure 7B is the chart of the tissue structure and pathological changes of each group at 4 weeks after the operation observed by hematoxylin-eosin (HE) and safranin-fast green (SO / FG) staining; Figure 7C is the chart of the tissue structure and pathological changes of each group at 8 weeks after the operation observed by hematoxylin-eosin (HE) and safranin-fast green (SO / FG) staining. DETAILED DESCRIPTION

[0056] The technical solutions in the present application will be described below with reference to the drawings.

[0057] In this document, the list of abbreviations, English and key terms is as follows:

[0058] UPy-Gel: urea-based pyrimidinone grafted gelatin; TA: tannic acid; MgO2: magnesium peroxide; FTIR: Fourier transform infrared spectroscopy; XPS: X-ray photoelectron spectroscopy; β-CD: β-cyclodextrin; EDC: 1-ethyl-(3-dimethylaminopropyl) carbodiimide; NHS: N-hydroxysuccinimide; GelMA: methacrylated gelatin; PBS: phosphate buffered saline; QUE: quercetin; GelMA-β-CD: β-cyclodextrin modified methacrylated gelatin; QUE@GelMA: quercetin-loaded β-cyclodextrin modified methacrylated gelatin composed core layer microneedle; TA@MgO2: metal phenolic network composed of tannic acid and magnesium peroxide; AF: annulus fibrosus; HA: hyaluronic acid; ADH: adipic acid dihydrazide; HA-ADH: hydrazide modified hyaluronic acid; HCA: dihydrocaffeic acid; HA-ADH-HCA: adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid; TA@MgO2@HA: shell layer microneedle composed of adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid loaded self-driven motor metal phenolic network composed of tannic acid and magnesium peroxide; TMH / QG MN: core-shell microneedle composed of core layer QUE@GelMA and shell layer TA@MgO2@HA; TBHP: tert-butyl hydroperoxide; MRI: magnetic resonance imaging; DHI: disc height index; H&E: hematoxylin & eosin staining; SO / FG: safranin O / fast green staining.

[0059] In this paper, the host-guest interaction in chemical reactions refers to the interaction between the host substance (or called the reactant) and the guest substance (or called the auxiliary agent) during the chemical reaction. Host-guest interaction describes a dynamic and reversible binding mode between molecules through non-covalent bonds (such as hydrogen bonds, van der Waals forces, hydrophobic interactions, etc.). The host molecule usually has a specific cavity structure (such as crown ether, cyclodextrin, etc.), which can selectively capture guest molecules (such as metal ions, small molecules, etc.) that match in size and shape.

[0060] Core-shell microneedles, as a new drug delivery system, have shown great application prospects due to their unique advantages such as layered loading, drug controlled release, minimally invasive penetration, and mechanical adaptation. To solve the problems existing in the prior art, the current research focus has shifted to the development of core-shell microneedles prepared using a core-shell domain loading strategy. The shell is made of high-hardness material to ensure the puncture ability, and the core is a porous sustained-release hydrogel that can load therapeutic components to match the mechanical properties of the annulus fibrosus. During IDD treatment, the core-shell microneedle can act as a patch to prevent further leakage of the nucleus pulposus, improve the microenvironment of the degenerated annulus fibrosus, and restore cell function, thereby promoting intervertebral disc repair.

[0061] Tannic acid (TA) is a natural polyphenol compound that can complex with ions and self-assemble into metal-phenolic networks (MPN) with metal ions. Metal phenolic is a new type of synthetic biomaterial with the ability to scavenge reactive oxygen species (ROS), which has great application potential in tissue engineering and has been widely concerned in ROS-related diseases. However, there is no report on designing a metal phenolic network to achieve acid microenvironment degradation, ROS scavenging, and environment-responsive self-driven oxygen release, and to alleviate hypoxia. Quercetin, also known as quercetin and quercetin, has a melting point of 314℃. It can be dissolved in ethanol, methanol, ethyl acetate, glacial acetic acid, pyridine, acetone, etc. It is not soluble in water, benzene, diethyl ether, chloroform, petroleum ether, etc. It is yellow in alkaline aqueous solution and almost insoluble in water. It tastes bitter.

[0062] The present application designs and prepares a new type of core-shell microneedle, which utilizes the asymmetric thrust force generated by the acid microenvironment of the annulus fibrosus to drive the motor movement, improves the hypoxic environment, and reduces the ROS and inflammation levels in the annulus fibrosus part, promotes the repair of the annulus fibrosus, and further realizes the restoration of the structure and function of the degenerative intervertebral disc. The present application designs and synthesizes a new type of core-shell microneedle, which loads a metal phenolic self-driven motor TA@MgO2 in the shell layer and a functional drug QUE in the core layer. By virtue of its time-sequential degradation characteristics, it utilizes the asymmetric thrust force generated by the acid microenvironment of the degenerative annulus fibrosus to drive the motor movement, improves the hypoxic environment, and reduces the ROS level; then it releases the functional drug to restore the cell vitality by improving the extracellular matrix microenvironment of the annulus fibrosus, thereby achieving the purpose of repairing the intervertebral disc. The time-sequential degradation of the microneedle of the present application is achieved through the core-shell structure. The shell layer first degrades when it contacts the acid microenvironment of the degenerative annulus fibrosus of the intervertebral disc, and then the core layer degrades, thereby achieving time-sequential degradation.

[0063] The advantages of the hyaluronic acid matrix modified by adipic acid dihydrazide-dihydrocaffeic acid in the shell layer are as follows: the ADH enhances the mechanical strength of the HA through amide bond crosslinking to withstand external puncture force and maintain structural stability, the pH responsiveness of the hydrazide group realizes dynamic response of the drug in the acid microenvironment of the annulus fibrosus; the phenolic hydroxyl group of HCA endows strong antioxidant and anti-inflammatory functions, which synergize with the mechanical support and pH sensing of ADH; HA, ADH and HCA maintain excellent biocompatibility and controllable degradability; at the same time, the multifunctional groups (carboxyl, hydrazide, phenolic hydroxyl) of the matrix efficiently load active ingredients through non-covalent / covalent interaction, and realize "stimulus-responsive controlled release" by means of pH response, finally constructing an integrated intelligent carrier of "structure strengthening-microenvironment regulation-biological activity-intelligent drug loading", which is suitable for biomedical scenarios that require precise microenvironment intervention.

[0064] The advantages of the methylacrylated gelatin GelMA-β-CD modified by β-cyclodextrin in the core layer are as follows: the β-CD in the methylacrylated gelatin GelMA-β-CD modified by β-cyclodextrin forms a host-guest inclusion with the drug molecule QUE through a hydrophobic cavity, significantly improves the drug loading efficiency and realizes slow release; and the ultraviolet light crosslinking characteristics of the GelMA and the controllable polymerization ability of the methylacryl group endow the material with adjustable mechanical properties and three-dimensional structure stability, which is suitable for complex tissue engineering scaffold construction; in addition, the hydrophilic surface of the β-CD and the GelMA cooperatively improve cell adhesion and proliferation, and the pH responsiveness of the β-CD can trigger precise release of the drug in an acidic microenvironment.

[0065] In order to make the technical problems, technical solutions and advantages of the present application clearer, specific embodiments will be described in detail below with reference to the drawings.

[0066] Example 1 Preparation of self-driven nanoparticle TA@MgO2

[0067] Tannic acid (TA) was dissolved in deionized water (concentration 10 mg / mL) and magnetically stirred (500 rpm, 25℃) until completely dissolved. Magnesium peroxide (MgO2) powder was added to anhydrous ethanol (concentration 20 mg / mL) and ultrasonically dispersed (power 200 W, 5 min) to a uniform suspension. The TA solution and MgO2 dispersion were mixed at a molar ratio and placed in an ice bath. The molar ratio of TA to MgO2 (1:1, 1:3, 1:5) was detected by Fourier infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS), respectively.

[0068] By ultrasonic-assisted complexation, different powers (200, 300, 400 W) and times (20, 30, 40 min) can be used. The reaction solution was centrifuged at 10,000 rpm for 10 min, the supernatant was discarded, and the precipitate was washed with deionized water for 3 times. The precipitate was placed in a vacuum drying box (40℃, 12 h) to obtain TA@MgO2 metal phenolic complex powder. The TA@MgO2 was detected by transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy and Zeta potential.

[0069] Experimental results:

[0070] The principle of imparting long-term self-driven characteristics to the nanomotor MgO2 by modifying MgO2 with tannic acid (TA) is as follows: the strong antioxidant capacity of TA can remove ROS generated by MgO2 catalyzing the decomposition of H2O2, prevent oxidative damage of the active site, and maintain its catalase-like activity; the complex structure of TA and MgO2 cooperatively consumes GSH and regenerates H2O2 through Fenton reaction, constructs a cascade self-energy supply cycle of “H2O2→O2+·OH→GSH consumption→H2O2 regeneration”, and maintains stable movement speed; in addition, the polyphenolic hydroxyl group of TA enhances the cell membrane adhesion and penetration ability, and inhibits the agglomeration of nanoparticles through Mg-O-C bond, prolonging the degradation half-life.

[0071] The experimental results of transmission electron microscopy, X-ray diffraction, X-ray photoelectron spectroscopy, and Zeta potential detection of the metal phenolic self-driven nanomotor TA@MgO2 are shown in Figures 1A-1F . Among them, Figure 1A is a transmission electron micrograph representing the surface morphology of the metal phenolic self-driven nanomotor TA@MgO2, wherein in order to impart long-term self-driven characteristics to the nanomotor, MgO2 is modified with tannic acid (TA) to synthesize the metal phenolic self-driven nanomotor TA@MgO2. The corresponding energy diffraction spectrum image proves that carbon (C), oxygen (O), and magnesium (Mg) elements are uniformly distributed in TA@MgO2.

[0072] Figure 1B is an X-ray diffraction (XRD) pattern analyzing the physical structure of MgO2 and TA@MgO2. As can be seen from Figure 1B , MgO2 has diffraction peaks corresponding to MgO2 (101), (110), and (112) near 37°, 42.8°, and 58.6°, respectively. TA@MgO2 also has diffraction peaks corresponding to MgO2 (101) and (002) near 36.7° and 25.6°, respectively. The above diffraction peaks indicate that the crystal faces of MgO2 appear in TA@MgO2. Among them, since TA does not form crystals, there is no diffraction peak.

[0073] Figure 1C is a graph of the Zeta potential of TA, MgO2, and TA@MgO2. Since TA is negatively charged, and Mg 2+ and the phenolic hydroxyl group of tannic acid form a dense cross-linked metal phenolic network covering the surface of MgO2 particles through coordination, the Zeta potential of TA@MgO2 is reduced from -6.9 mV to -18.68 mV compared with MgO2.

[0074] Figures 1D-1F is a graph of X-ray photoelectron spectroscopy (XPS) used to further confirm the chemical composition of TA@MgO2. As Figure 1DAs shown, TA@MgO2 contains magnesium (Mg) and oxygen (O) elements, respectively, with Mg as the main component. 2+ and peroxide (O2) 2- The presence of magnesium peroxide (in the form of ) indicates the presence of magnesium peroxide. For example... Figure 1E As shown, a peroxide (OO) fingerprint peak at 530.98 eV, belonging to TA@MgO2, appears, distinct from the characteristic peak of the Mg-O bond at ~530.5 eV, thus excluding MgO interference. Meanwhile, as... Figure 1F As shown, a characteristic peak belonging to Mg 1s in TA@MgO2 appears at 1303.96 eV. Since divalent magnesium in TA@MgO2 is in an electron-rich environment, its binding energy is slightly higher than that of Mg 1s in Mg-O (~1303.8 eV), indicating that high-purity TA@MgO2 has been successfully synthesized.

[0075] Example 2: Performance Testing of TA@MgO2

[0076] Experimental steps:

[0077] (1) DPPH free radical scavenging experiment:

[0078] Different concentrations of TA@MgO2 were thoroughly mixed with DPPH solution (0.2 mmol / L, Beijing Solarbio Science & Technology Co., Ltd.) in a light-protected environment at 37°C. Color changes of the pure DPPH solution and the sample-DPPH mixture were observed after one day. The absorbance at 517 nm was measured using a UV spectrophotometer. The preparation of TA@MgO2 can be found in Example 1. To further quantify the antioxidant capacity, the absorbance of the pure DPPH solution and the sample-DPPH mixture at 517 nm was measured using a microplate reader. The DPPH free radical scavenging capacity was calculated using the following formula:

[0079] .

[0080] Where Ac is the absorbance of the pure DPPH solution at 517 nm, and As is the absorbance of the sample and DPPH mixture at 517 nm.

[0081] (2) SOD enzyme activity detection experiment:

[0082] To detect the SOD-like catalytic activity of the sample, different concentrations of TA@MgO2 were mixed with the detection solution (total SOD activity detection kit, Shanghai Biyun Tian Biotechnology Co., Ltd.) at room temperature for 20 minutes. Then the substrate working solution and enzyme working solution (total SOD activity detection kit, Shanghai Biyun Tian Biotechnology Co., Ltd.) were added, and the reaction system was finally collected. The absorbance value (A) at 450 nm wavelength was detected by an enzyme marker, and the SOD enzyme activity of different concentrations of TA@MgO2 was calculated based on the inhibition rate. The calculation formula is: inhibition percentage = [(A 空白对照1 -A 空白对照2 ) - (A 样品 -A 空白对照3 )] / (A 空白对照1 -A 空白对照2 ) x 100%, SOD enzyme activity unit in the sample to be tested = SOD enzyme activity unit in the detection system = inhibition percentage / (1 - inhibition percentage) units (the calculation formula is according to the instruction manual of the detection kit).

[0083] (3) H2O2 scavenging experiment:

[0084] 50 μL of different concentrations of TA@MgO2 were mixed with 100 μM H2O2 detection reagent (hydrogen peroxide detection kit, Shanghai Biyun Tian Biotechnology Co., Ltd.), and incubated at room temperature for 1 day. The absorbance of each group of samples at 560 nm wavelength was detected by an enzyme marker. The H2O2 scavenging ability of different concentrations of TA@MgO2 was calculated according to the standard curve.

[0085] Experimental results:

[0086] The experimental results are shown in Figures 2A-2D .

[0087] Among them, Figure 2A is the DPPH free radical scavenging experiment result graph of the self-driven nanomotor TA@MgO2. From Figure 2A , it can be seen that with the increase of the sample concentration from 0.1 mg / mL to 10 mg / mL, the DPPH scavenging ability of TA@MgO2 increases significantly, so the color of the sample and DPPH mixed solution gradually changes from purple to yellow with the increase of TA@MgO2 concentration, and the corresponding absorption peak at 517 nm gradually decreases.

[0088] Figure 2B is the graph of the DPPH scavenging ability of the self-driven nanomotor TA@MgO2. From Figure 2BIt can be seen that the ability of TA@MgO2 to scavenge DPPH gradually increases with the increase of sample concentration, in which the ability of 5 mg / mL and 10 mg / mL TA@MgO2 to scavenge DPPH is 1.81 times and 1.91 times of that of 0.1 mg / mL sample respectively, indicating that TA@MgO2 can effectively scavenge DPPH free radicals.

[0089] Figure 2C is a graph reflecting the SOD-like activity of TA@MgO2. The experimental results show that the SOD-like activity of TA@MgO2 gradually increases with the increase of sample concentration, and the average SOD inhibition rate of 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL and 10 mg / mL TA@MgO2 is 30.6%, 45.7%, 60.2%, 73.5% and 77.4% respectively, and the corresponding SOD-like activity is 0.44 unit, 0.84 unit, 1.51 unit, 2.77 unit and 3.42 unit respectively according to the formula. The results show that TA@MgO2 can effectively scavenge active oxygen by catalyzing the dismutation reaction of superoxide anion free radicals (O2 - ) to generate oxygen and hydrogen peroxide, and exhibits SOD-like antioxidant activity.

[0090] Figure 2D is a graph of the H2O2 scavenging ability of TA@MgO2. It can be seen from Figure 2D that TA@MgO2 exhibits high H2O2 scavenging ability, and the H2O2 scavenging ability gradually increases with the increase of sample concentration. After co-incubation for 1 day, the H2O2 scavenging ability of 10 mg / mL TA@MgO2 group increased by 16.6% to 30.91% compared with that of 0.1 mg / mL TA@MgO2 group, confirming that TA@MgO2 can quickly scavenge H2O2.

[0091] Example 3 Assembly of core-shell microneedle TMH / QG-MN

[0092] (1) Synthesis of core layer material QUE@GelMA

[0093] Dissolve 1 g of β-cyclodextrin (β-CD) in 50 mL of deionized water, add 0.5 mmol of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 0.5 mmol of NHS (N-hydroxysuccinimide), magnetic stirring (500 rpm, 25°C, 2 h) to obtain carboxyl-activated β-CD. Dissolve 2 g of methacrylated gelatin (GelMA, purchased from Suzhou Yongqinquan Intelligent Equipment Co., Ltd.) in 50 mL of phosphate buffer (PBS), add the activated β-CD solution, continue stirring (37°C, 12 h). Transfer the mixture to a dialysis bag (molecular weight cut-off 8-14 kDa), dialyze in deionized water for 3 days (6 times of water change), remove unreacted EDC / NHS and free β-CD. Freeze-drying to obtain β-CD modified GelMA (GelMA-β-CD). Dissolve 50 mg of quebrachitol (QUE) in 10 mL of anhydrous ethanol, slowly add to the GelMA-β-CD solution (5% w / v, DPBS), stir at 37°C for 30 min to form an inclusion complex solution, centrifuge at 12000 rpm for 10 min, collect the supernatant, vacuum freeze-drying to obtain QUE@GelMA.

[0094] (2) Synthesis of shell material TA@MgO2@HA

[0095] To match the mechanical needs of the annulus fibrosus (AF), hydrazine groups of adipic acid dihydrazide (ADH) were introduced into hyaluronic acid (HA) to enhance the cross-linking ability of HA, thereby improving the mechanical strength and stability of the microneedle. The specific steps are as follows: accurately weigh 1 g of hyaluronic acid (HA, 100 kDa) and dissolve it in 100 mL of pure water, add 2.3 g of EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) and 4 g of adipic acid dihydrazide (ADH), and magnetically stir (500 rpm, 25°C) for 2 h to activate the β-carboxyl group. Then adjust the pH to 6.8 with HCl and react at room temperature for 6 h to complete the hydrazine modification, obtaining crude hydrazinyl hyaluronic acid (HA-ADH). Transfer the crude product to a dialysis bag with a molecular weight cut-off of 800 Da, and dialyze in deionized water for 48 h (1 water change every 12 h) to remove unreacted EDC and ADH, and freeze-dry (-50°C, 10 Pa) for 12 h to obtain pure HA-ADH. Existing HA-ADH in the prior art can also be used, for example, which can be purchased from Xi'an Ruishi Biological Co., Ltd.

[0096] Take HA-ADH (0.5 g) dissolved in 50 mL PBS (pH 7.4), add 1.2 g dihydrocaffeic acid (HCA), ultrasonic dispersion (200 W, ice bath) for 30 min to make it uniformly dispersed; then add 0.8 g EDC and 0.4 g NHS (N-hydroxysuccinimide), adjust the pH to 6.5, room temperature magnetic stirring (500 rpm) reaction for 12 h, through amide bond coupling to make HCA covalently modified on the hydrazine group of HA-ADH. After the reaction is completed, the mixed solution is transferred to a dialysis bag with a molecular weight cut-off of 3.5 kDa, dialyzed with PBS for 48 h (every 12 h change 1 time) to completely remove unreacted EDC, NHS and free HCA, and finally vacuum freeze-dried (-50℃, 10Pa) for 48h to obtain adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid (HA-ADH-HCA).

[0097] TA@MgO2 powder is added to the HA-ADH-HCA solution (pH 7.4, concentration 5% w / v) at a mass ratio of 1:10 (TA@MgO2:HA-ADH-HCA). The mixed solution is treated with an ultrasonic disperser (power 200 W, ice bath condition) for 20 min to ensure that TA@MgO2 is uniformly dispersed in the HA-ADH-HCA matrix.

[0098] The mixed solution is injected into the mold, pre-frozen at -80℃ for 6 h, freeze-dried for 24 h to form a porous composite scaffold (here, the porous composite scaffold is defined based on the SEM results, and the step of freeze-drying here increases the contact area between TA@MgO2 and HA-ADH-HCA, thereby improving the reaction efficiency of the two and accelerating oxygen release), then soaked in deionized water (30 min, 3 times) to remove unbound TA@MgO2, and freeze-dried again to obtain TA@MgO2@HA composite. +

[0099] (3) Preparation of core-shell microneedle TMH / QG MN

[0100] The solution of the shell material TA@MgO2@HA (concentration 20% w / v) is injected into the mold (PDMS microneedle mold), vacuum dried, then the solution of the core material QUE@GelMA (concentration 20% w / v) is added, followed by vacuum drying and demolding, and finally the desired core-shell microneedle TMH / QG MN is obtained. The steps of preparing the core-shell microneedle can also be seen in Example 4, for example.

[0101] (4) Measurement of the nuclear magnetic resonance hydrogen spectrum of hydrazine (ADH) modified HA, hydrazine (ADH)-dihydrocaffeic acid (HCA) modified HA (HA-ADH-HCA), and β-cyclodextrin (β-CD) modified GelMA (GelMA-β-CD). ​

[0102] Experimental results:

[0103] The experimental results are shown in Figures 3A-3E

[0104] wherein, Figure 3A is a diagram of the chemical synthesis steps of hyaluronic acid (HA) modified with hydrazide (ADH) - dihydrocaffeic acid (HCA) (HA-ADH-HCA). Figure 3B is a diagram of the chemical synthesis steps of gelatin methacryloyl (GelMA) modified with β-cyclodextrin (β-CD) (GelMA-β-CD). Figure 3C is a graph of the nuclear magnetic resonance hydrogen spectrum of HA modified with hydrazide (ADH). The results show that the product after reaction appears a new peak representing the ADH methylene at 2.3 ppm, indicating that HA-ADH is successfully synthesized. Figure 3D is a graph of the nuclear magnetic resonance hydrogen spectrum of HA modified with hydrazide (ADH) - dihydrocaffeic acid (HCA) (HA-ADH-HCA). The results show that the product after reaction appears a methylene peak representing ADH at 2.46 ppm, an α-methylene peak representing HCA at 2.61 ppm, a β-methylene peak representing HCA at 2.81 ppm, and a HCA benzene ring proton peak at 6.75 ppm, confirming that HA-ADH-HCA is successfully synthesized. Figure 3E is a graph of the nuclear magnetic resonance hydrogen spectrum of GelMA modified with β-cyclodextrin (β-CD) (GelMA-β-CD). The results show that the methyl peak, the CD cavity proton peak, the CD anomeric proton peak and the cis olefinic hydrogen peak of MA appear at 1.82 ppm, 3.63 ppm, 5.14 ppm and 5.28 ppm, respectively, indicating that GelMA-β-CD is successfully synthesized.

[0105] Example 4

[0106] Before injecting the core layer material QUE@GelMA (dissolved in DPBS buffer) prepared in Example 3 into the PDMS microneedle mold, the PDMS mold was first subjected to plasma cleaning to reduce the surface energy and enhance the hydrophilicity, and then the inner wall of the mold was wetted with deionized water (to avoid strong adhesion of the material to the mold). The QUE@GelMA solution was slowly injected into the mold at a flow rate of 0.5 mL / min, ensuring that the solution completely filled the microchannels and no air bubbles were left; after injection was completed, the mold was placed in a vacuum drying oven for 4 hours to allow the solvent (DPBS) to fully evaporate, and the material formed a solid microneedle structure. After drying was completed, the edge of the mold was gently pressed with a wet cotton swab, and the microneedles were slowly peeled off in the vertical direction to obtain the core layer microneedles QUE@GelMA MN.

[0107] ​Similarly, the preparation process of the shell layer material TA@MgO2@HA (dissolved in PBS buffer) microneedle is consistent with the core layer: after the PDMS mold is subjected to the same plasma cleaning and wetting treatment, the TA@MgO2@HA solution is injected, and after vacuum drying, the wet cotton swab is peeled off to obtain the shell layer microneedle TA@MgO2@HA MN.

[0108] The assembly process of the core-shell structure microneedle TMH / QG MN strictly controls the interface bonding: first, the TA@MgO2@HA MN is re-fixed in the PDMS mold (to ensure that the shell layer is centered), and then it is placed in a vacuum drying box for pre-drying for 1 hour to enhance the bonding force between the shell layer and the mold; then, the core layer material QUE@GelMA solution is slowly added to the microchannel on the surface of the shell layer microneedle at a flow rate of 0.3 mL / min (to avoid damaging the shell layer structure), and it is ensured that the core layer completely fills the hollow structure inside the shell layer. After the addition is completed, vacuum drying is continued for 3 hours to enable the core layer and the shell layer to be tightly bonded through intermolecular forces; finally, the microneedle edge is lightly clamped with a wet tweezers, and it is peeled off along the normal direction of the mold at a uniform speed to obtain the core-shell structure microneedle TMH / QG MN.

[0109] 1. Fluorescence microscope photographs of the core layer microneedle QUE@GelMA MN (green fluorescence), the shell layer microneedle TA@MgO2@HA MN (red fluorescence), and the core-shell microneedle TMH / QG MN are taken;

[0110] 2. The core-shell microneedle TMH / QG MN is used to puncture pig skin, and the puncture condition is recorded by taking photographs, and the skin slice is subjected to hematoxylin & eosin (H&E) staining.

[0111] 3. Degradation behavior experiment of the core-shell microneedle TMH / QG MN:

[0112] The core-shell microneedle TMH / QG MN is placed in a normal physiological microenvironment (pH 7.4), an acidic microenvironment (pH 6.5), a reactive oxygen species (ROS) rich microenvironment, and a degenerated annulus fibrosus microenvironment (pH 6.5+ROS) for 14 days. The core-shell microneedle TMH / QG MN is subjected to microscope photographing.

[0113] Experimental results:

[0114] The experimental results are shown in Figures 4A-4C .

[0115] Among them, Figure 4A is the fluorescence microscope image of the core layer microneedle QUE@GelMA MN (green fluorescence), the shell layer microneedle TA@MgO2@HA MN (red fluorescence), and the core-shell microneedle TMH / QG MN, from Figure 4AAs can be seen, the outer layer of the core-shell microneedle TMH / QG MN exhibits red fluorescence, while the core shows green fluorescence, proving that the core-shell structure was successfully prepared.

[0116] Figure 4B This image shows a section of porcine skin punctured with a core-shell microneedle (TMH / QG MN) and stained with hematoxylin and eosin (H&E). From... Figure 4B It can be seen that TMH / QG MN can effectively penetrate dense tissues such as porcine skin. The prepared microneedles have a height of 500-3000 μm (e.g., 800 μm), and the puncture depth can reach the inner layer of the annulus fibrosus. These results demonstrate that the core-shell microneedles TMH / QGMN have excellent puncture capabilities, exhibiting the potential to puncture and fix to the annulus fibrosus to exert a therapeutic effect.

[0117] Figure 4C This is a graph depicting the degradation behavior of core-shell microneedles TMH / QG MN. TMH / QG MN was placed in normal physiological microenvironments (pH 7.4), acidic microenvironments (pH 6.5), reactive oxygen species (ROS)-rich microenvironments, and degenerated fibrous ring microenvironments (pH 6.5 + ROS) for 14 days. The pH 7.4 + H2O2 setting represents the degradation under purely ROS-rich conditions, while the pH 6.5 + H2O2 setting represents the degradation under pH 6.5 + ROS conditions. Figure 4C It can be found that the core-shell microneedles TMH / QG MN have good stability and appropriate degradation rate in four microenvironments. This ensures that the stable structure required for early treatment can be used to fix the annulus fibrosus, while avoiding long-term residence that restricts the regeneration of the annulus fibrosus tissue.

[0118] Example 5

[0119] The core-shell microneedles TMH / QG MN from Example 1 were placed in liquids simulating normal physiological microenvironments (pH 7.4), acidic microenvironments (pH 6.5), reactive oxygen species (ROS)-rich microenvironments, and degenerated fibrous ring microenvironments (pH 6.5 + ROS) for 14 days. The supernatants were then collected, and the release of tannic acid (TA) and quercetin (QUE) was measured. The method for detecting the release of tannic acid and quercetin was as follows: the absorbance of the supernatant at A260 was measured using a UV spectrophotometer.

[0120] Experimental results:

[0121] Experimental results are as follows Figures 5A-5B As shown.

[0122] Figure 5AFigure 6 is a graph showing the release of tannic acid (TA) from core-shell microneedle TMH / QG MN in normal physiological microenvironment (pH 7.4), acidic microenvironment (pH 6.5), active oxygen (ROS) rich microenvironment, and degenerative annulus fibrosus microenvironment (pH 6.5+ROS) for 14 days. Figure 5A The results show that the rate of TA release from core-shell microneedle TMH / QG MN in the degenerative annulus fibrosus microenvironment (pH 6.5+ROS) is much higher than that in the other three microenvironments, and the amount of TA released reaches 124 μg after 14 days of co-culture, indicating that core-shell microneedle TMH / QG MN can timely and continuously release TA in the degenerative annulus fibrosus microenvironment to reduce the inflammation level at the annulus fibrosus site, thereby exerting the potential of therapeutic effect.

[0123] Figure 5B Figure 7 is a graph showing the release of quercetin (QUE) from core-shell microneedle TMH / QG MN in normal physiological microenvironment (pH 7.4), acidic microenvironment (pH 6.5), active oxygen (ROS) rich microenvironment, and degenerative annulus fibrosus microenvironment (pH 6.5+ROS) for 14 days. Figure 5B The results show that at the same time point, the amount of QUE released from core-shell microneedle TMH / QG MN in the degenerative annulus fibrosus microenvironment (pH 6.5+ROS) is significantly higher than that in the other three microenvironments, and the amount of QUE released reaches 36 μg after 14 days of co-culture, indicating that core-shell microneedle TMH / QG MN can timely and continuously release QUE in the degenerative annulus fibrosus microenvironment to reduce the ferroptosis of annulus fibrosus cells, thereby exerting the potential of therapeutic effect.

[0124] Example 6 Treatment effect 1: Intracellular reactive oxygen species level test of annulus fibrosus cells co-cultured with core-shell microneedle, shell microneedle and core-shell microneedle for 48 hours under oxidative stress induction

[0125] Primary annulus fibrosus cells were extracted from rat annulus fibrosus tissue. After the addition of the oxidative stress inducer tert-butyl hydroperoxide (TBHP) (100 μM), the rat primary annulus fibrosus cells were co-cultured with the core-shell microneedle TMH / QG MN (100 mg) for two days, and then the intracellular reactive oxygen species (ROS) level was determined using the reactive oxygen species detection kit produced by Shanghai Biyun Tian Biotechnology Co., Ltd. The experimental steps were performed according to the kit instructions. Among them, the experimental steps are as follows: take the logarithmic growth period annulus fibrosus cells, inoculate in the 24-well plate containing cover glass, and culture to 70%-80% fusion; discard the old culture medium, and gently wash the cells with PBS, add serum-free medium containing 100 μM TBHP, and incubate for 2 h; discard the TBHP solution, and gently wash with PBS, then add QUE@GelMA MN, TA@MgO2@HA MN, and TMH / QG MN microneedles according to the grouping, add serum-free medium, and co-culture at 37°C, 5% CO2 for 48 h; discard the culture medium, and gently wash with PBS, add DCFH-DA probe (10 μM), and incubate in the dark for 30 min; wash off the free probe with PBS, resuspend the cells with PBS, and observe and collect fluorescence images under an inverted fluorescence microscope.

[0126] Experimental results:

[0127] The experimental results are shown in Figure 6 . Figure 6 is a graph of the intracellular reactive oxygen species (ROS) level of the annulus fibrosus cells co-cultured with the core-shell microneedle TMH / QG MN for two days after the addition of the oxidative stress inducer tert-butyl hydroperoxide (TBHP). As can be observed from Figure 6 , compared with the control group, the ROS level in the annulus fibrosus cells of the simple TBHP group is obviously increased, and a large number of green fluorescence appears. Although the core-shell microneedle QUE@GelMA MN and the shell layer microneedle TA@MgO2@HA MN can both reduce the ROS level (i.e., the green fluorescence is reduced relative to the TBHP group), the effect is not as good as that of the core-shell microneedle TMH / QG MN. This is because the core-shell microneedle TMH / QG MN not only carries TA that consumes ROS to directly reduce the ROS level, but also carries the drug QUE that can improve cell function and reduce ROS production. The above results suggest that the core-shell microneedle TMH / QG MN can reduce the intracellular ROS level of degenerative annulus fibrosus cells through synergistic action at the cell level and avoid cell oxidative stress.

[0128] Example 7: Treatment effect 2 - treatment effect of core-shell microneedle TMH / QG MN after rat tail vertebra intervertebral disc puncture

[0129] Experimental procedure:

[0130] Adult male SD rats (purchased from Chengdu Enswell Biological Technology Co., Ltd.) (body weight 250-300 g) were randomly divided into 3 groups: sham operation group (Sham group or control group), acupuncture group (Model group or intervertebral disc degeneration group), acupuncture + core-shell microneedle group or (Core / Shell group or core-shell microneedle treatment group), 10 rats in each group.

[0131] 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. Without any treatment, it was the intervertebral disc degeneration model group (Model group or intervertebral disc degeneration group).

[0132] The experimental group (Core / Shell group or core-shell microneedle treatment group) implanted core-shell microneedle TMH / QG MN at the annulus fibrosus injury site. The tail vertebrae were taken out for imaging examination and sectioning for histological evaluation at 4 and 8 weeks after operation. Among them, the imaging examination experimental procedure: isoflurane inhalation anesthesia was used to anesthetize the rats, the rat tail vertebrae were stretched out and placed in the center of the X-ray machine tray, the tail was padded with a thin cotton pad below to maintain the natural curvature, and was lightly bound with tape. Adjust the X-ray machine parameters (voltage 40-50 kVp, current 5-10 mA, exposure time 0.1-0.5 s), focus on the tail vertebrae area, complete single exposure quickly; then use small animal magnetic resonance to take T2 weighted image;

[0133] Histological evaluation procedure: The target segment of the coccyx was taken and fixed in 4% paraformaldehyde (4℃) for 24-48 h; after fixation, it was dehydrated by gradient ethanol (70% ethanol 2 h → 80% ethanol 2 h → 95% ethanol I 2 h → 95% ethanol II 2 h → 100% ethanol I 1 h → 100% ethanol II 1 h), and then cleared with xylene (clearing I 1 h → clearing II 1 h); the tissue block was immersed in paraffin (60℃ constant temperature oven) overnight, and 5 μm continuous sections were made along the long axis of the coccyx using a paraffin microtome, mounted on poly-L-lysine-treated glass slides, and fixed by baking at 60℃ for 2 h. After dewaxing and rehydration, sections were stained with hematoxylin and eosin (HE): staining with hematoxylin (1%) for 5-8 min, rinsing with tap water to remove excess stain; differentiating with hydrochloric acid ethanol (1%) for 3-5 s (cell nuclei were clearly blue under the microscope), then re-blued with tap water for 10 min; staining with eosin (0.5% alcohol-soluble) for 1-3 min, followed by gradient ethanol dehydration (70%→80%→95%→100%, 30 s each), clearing twice with xylene (5 min each time), and mounting with neutral resin. Simultaneous safranin and fast green staining: After dewaxing and rehydration, stain with safranin (1% acid red) for 30-60 min (to fully visualize the cartilage matrix and nucleus pulposus proteoglycans), then rinse with tap water; stain with fast green (0.1%) for 10-15 s (to specifically stain collagen fibers), then rinse with tap water to stop the reaction; dehydrate with a gradient of ethanol (70%→80%→95%→100%, 30 s each), clear twice with xylene (5 min each time), and mount with neutral resin. After staining, observe using an optical microscope (10×, 20×, 40× objectives).

[0134] Experimental results:

[0135] Experimental results are as follows Figures 7A-7C As shown.

[0136] Figure 7A These are images of X-ray and MRI images of the rat caudal vertebrae at 4 and 8 weeks post-surgery. The dashed boxes indicate the experimentally observed segments. From Figure 7A As can be seen from the data, compared with the Model group, the Core / Shell group can effectively maintain intervertebral height, prevent nucleus pulposus leakage and degradation, and maintain the water content of the nucleus pulposus.

[0137] Figure 7B These are images showing the tissue structure and pathological changes in each group 4 weeks post-surgery, observed using hematoxylin and eosin (HE) and safranin-fast green (SO / FG) staining. Figure 7B As can be seen, the annulus fibrosus structure in the Model group was disordered, and the boundary between the nucleus pulposus and the annulus fibrosus disappeared. In contrast, the annulus fibrosus tissue in the Core / Shell group was neatly arranged, and the boundary between the nucleus pulposus and the annulus fibrosus was clear.

[0138] Figure 7CThe figures of the histological structure and pathological changes of each group at 8 weeks after operation were observed by hematoxylin-eosin (HE) and safranin O / fast green (SO / FG) staining. Figure 7C It can be seen that the boundary of nucleus pulposus and annulus fibrosus of the Model group disappeared. In contrast, the tissue arrangement of the annulus fibrosus part of the Core / Shell group was neat, and the boundary of the nucleus pulposus and the annulus fibrosus was clear.

[0139] The above results show that the nucleus shell microneedle TMH / QG MN can effectively promote the repair of the annulus fibrosus, maintain the intervertebral disc tissue structure, and thus inhibit the intervertebral disc degeneration after the intervertebral disc puncture of the rat tail vertebra.

[0140] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited to this. Any person skilled in the art can easily think of changes or replacements within the technical range disclosed by the present application, which should be covered within the protection scope of the present application. Therefore, the protection scope of the present application should be subject to the protection scope of the claims.

Claims

1. A core-shell microneedle for treating intervertebral disc degeneration, the core-shell microneedle comprising a base and a plurality of microneedles disposed on the base, each microneedle having a core-shell structure, the core-shell structure comprising a shell layer and a core layer, characterized in that, The shell comprises a metal phenolic self-driven nanomotor and hyaluronic acid modified with adipic acid dihydrazide-dihydrocaffeic acid. The metal phenolic self-driven nanomotor is a metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide. The chemical formula of the hyaluronic acid modified with adipic acid dihydrazide-dihydrocaffeic acid is shown below: ， Wherein, n is 500-2500, and the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide is dispersed in the hyaluronic acid modified with adipic acid dihydrazide-dihydrocaffeic acid; and The core layer comprises a porous sustained-release hydrogel, which loads the drug quercetin through host-guest interaction.

2. The core-shell microneedle according to claim 1, characterized in that, The metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide is formed by the following method: Mg in a magnesium peroxide suspension 2+ A dense cross-linked metal phenolic network is formed by coordination with the phenolic hydroxyl groups of tannic acid and covers the surface of MgO2 particles, thus obtaining a metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide. Alternatively, the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide can be prepared as follows: a tannic acid solution is mixed with a magnesium peroxide suspension, and a complexation reaction is carried out with ultrasonic assistance to obtain the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide.

3. The core-shell microneedle according to claim 1, characterized in that, The adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid is prepared by the following method: adding dihydrocaffeic acid (HCA) to the adipic acid dihydrazide-modified hyaluronic acid (HA-ADH) solution, and reacting to obtain the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid (HA-ADH-HCA), the chemical formula of which is shown below: ； And / or, the mass ratio of the metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide to the hyaluronic acid modified with adipic acid dihydrazide-dihydrocaffeic acid is 1:5-1:10, or 1:

10.

4. The core-shell microneedle according to claim 1, characterized in that, The porous sustained-release hydrogel is β-cyclodextrin-modified methacrylamide gelatin GelMA-β-CD; And / or, the β-cyclodextrin-modified methacrylamide gelatin GelMA-β-CD is prepared by the following method: a) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to a β-cyclodextrin solution and react to obtain carboxyl-activated β-cyclodextrin; b) Add the carboxyl-activated β-cyclodextrin to the methacrylated gelatin solution and react to obtain the β-cyclodextrin-modified methacrylated gelatin GelMA-β-CD, the chemical reaction formula of which is shown below: ； And / or, the β-cyclodextrin-modified methacrylamide gelatin GelMA-β-CD forms an inclusion complex with quercetin through host-guest interaction, wherein the mass ratio of quercetin to the β-cyclodextrin-modified methacrylamide gelatin GelMA-β-CD is 1:1-1:5, or 1:

2.

5. The method for preparing core-shell microneedles according to any one of claims 1-4, characterized in that, The method includes: (1) Preparation of a metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide: Tannic acid solution was mixed with magnesium peroxide suspension, and a complexation reaction was carried out with ultrasonic assistance to obtain a metal phenolic complex network TA@MgO2 composed of tannic acid and magnesium peroxide. (2) Synthesis of shell material TA@MgO2@HA: The TA@MgO2 obtained in step (1) is added to a hyaluronic acid solution modified with adipic acid dihydrazide-dihydrocaffeic acid, and the mixture is treated with ultrasound to make TA@MgO2 uniformly dispersed, thereby obtaining the shell material TA@MgO2@HA; (3) Synthesis of core layer materials: The core layer material includes a porous sustained-release hydrogel, which loads the drug quercetin through host-guest interaction; (4) Assemble into core-shell microneedles: The shell material TA@MgO2@HA is injected into a mold, dried, and then a solution of the core material is added. After drying, the mold is removed to obtain the core-shell microneedles.

6. The preparation method according to claim 5, characterized in that, In step (1), The molar ratio of tannic acid to magnesium peroxide is 1:1-1:5, or 1:3; And / or, the power of the ultrasound is 200 W-400 W, or 200 W; And / or, the duration of the ultrasound is 20-40 min, or 40 min.

7. The preparation method according to claim 5, characterized in that, In step (2), The mass ratio of TA@MgO2 to the hyaluronic acid modified with adipic acid dihydrazide-dihydrocaffeic acid is 1:5-1:10, or 1:

10. And / or, the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid is prepared by the following method: adding dihydrocaffeic acid (HCA) to the adipic acid dihydrazide-modified hyaluronic acid (HA-ADH) solution, reacting to obtain hydrazide-dihydrocaffeic acid double-modified hyaluronic acid (HA-ADH-HCA), the chemical formula of which is shown below: 。 8. The preparation method according to claim 6, characterized in that, In step (3), The porous sustained-release hydrogel is β-cyclodextrin-modified methacrylamide gelatin (GelMA-β-CD). And / or, the β-cyclodextrin-modified methacrylamide gelatin GelMA-β-CD is prepared by the following method: a) Add 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide to a β-cyclodextrin solution and react to obtain carboxyl-activated β-cyclodextrin; b) Add the carboxyl-activated β-cyclodextrin to the methacrylated gelatin solution and react to obtain the β-cyclodextrin-modified methacrylated gelatin GelMA-β-CD, the chemical formula of which is shown below: ； And / or, an anhydrous ethanol solution of quercetin is mixed with a β-cyclodextrin-modified methacrylamide gelatin GelMA-β-CD solution to form an inclusion complex solution, thereby obtaining the core layer material QUE@GelMA; And / or, the mass ratio of the quercetin to the β-cyclodextrin-modified methacrylamide gelatin GelMA-β-CD is 1:1 to 1:5, or 1:

2.

9. A hyaluronic acid modified with adipicohydrazide-dihydrocaffeic acid, characterized in that, The chemical formula of the adipic acid dihydrazide-dihydrocaffeic acid modified hyaluronic acid is shown below: ， Where n is between 500 and 2500.

10. The method for preparing hyaluronic acid modified with adipic acid dihydrazide-dihydrocaffeic acid according to claim 9, characterized in that, The preparation method includes: adding dihydrocaffeic acid (HCA) to the adipic acid dihydrazide-modified hyaluronic acid (HA-ADH) solution, reacting to obtain the adipic acid dihydrazide-dihydrocaffeic acid-modified hyaluronic acid (HA-ADH-HCA), the chemical formula of which is shown below: 。

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