A traditional Chinese medicine plaster for lumbar intervertebral disc herniation and a preparation method thereof

Abstract

The application discloses a traditional Chinese medicine plaster for lumbar disc herniation and a preparation method thereof, and relates to the technical field of biological medicine. In the microneedle layer, macromolecular antler active components are loaded, and a liposome transition depot layer and an enzyme-responsive hydrogel controlled-release layer are constructed on the surface of the microneedle layer. Through the system integration of the three-layer structure, a complete treatment chain of puncture, transdermal penetration, transition depot, intelligent response and controlled release is constructed. The microneedle layer breaks through the physical barrier of the stratum corneum, directly delivers the antler active components and analgesic anti-inflammatory components to the dermis layer, and realizes rapid effect. The liposome transition depot layer provides secondary release and protection, maintains the drug concentration platform, and realizes continuous supply. The enzyme-responsive hydrogel controlled-release layer intelligently adjusts the drug release rate according to the pathological state of the lesion, and realizes on-demand drug release. The antler active components promote the repair of nucleus pulposus cells and nerve regeneration, and the CeO2@PDA nanoenzyme removes ROS to improve the microenvironment, realizing the treatment of both the symptoms and the root cause.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically, to a traditional Chinese medicine plaster for lumbar disc herniation and its preparation method. Background Technology

[0002] Lumbar disc herniation is a common degenerative spinal disease in clinical practice. Its main pathological basis includes intervertebral disc degeneration, nucleus pulposus protrusion, mechanical compression of nerve roots, and inflammatory responses induced by the protruding nucleus pulposus tissue. Patients often present with symptoms such as low back pain, radiating pain in the lower extremities, numbness, and limited mobility. In addition to mechanical compression, pathological processes such as the release of inflammatory factors, abnormal expression of matrix metalloproteinases, elevated levels of reactive oxygen species, and degradation of the extracellular matrix of the nucleus pulposus cells are also considered to be closely related to persistent pain, tissue degeneration, and nerve root stimulation. Therefore, local treatment for lumbar disc herniation needs not only to achieve rapid analgesia and anti-inflammation but also to consider the regulation of the degenerative microenvironment and support for tissue repair.

[0003] Currently, commonly used clinical treatments include oral nonsteroidal anti-inflammatory drugs (NSAIDs), local injections, physical therapy, traction, and surgery. While oral medications can relieve pain and inflammation, they can cause gastrointestinal irritation, liver and kidney burden, and systemic adverse reactions. Invasive treatments such as local injections require precise manipulation and are not suitable for long-term or frequent use. Surgical treatments are typically suitable for patients who do not respond to conservative treatment or have severe nerve compression. In contrast, topical medications such as plasters, gels, and poultices offer advantages such as ease of use, better patient compliance, and lower systemic exposure, making them widely used as adjunctive treatments for low back and leg pain and chronic soft tissue pain.

[0004] However, existing topical preparations still have significant shortcomings. Traditional Chinese medicine plasters are mostly made by simply mixing medicinal powders, alcohol extracts, or volatile oil components with a base, relying mainly on passive diffusion of the drug through the skin to exert its effects. The stratum corneum, as the main transdermal barrier, has a significant blocking effect on hydrophilic drugs and large molecular active ingredients, making it difficult for many potentially repairing active substances to effectively penetrate into the deeper layers of the skin. In particular, the active components such as polypeptides and growth factors contained in deer antler usually have strong hydrophilicity and large molecular weight, resulting in low transdermal efficiency when applied directly, making it difficult to fully exert their effects of promoting tissue repair, regulating inflammation, and neuroprotection.

[0005] Deer antler is a traditional and precious Chinese medicine containing various active ingredients such as polypeptides, proteins, growth factors, amino acids, and polysaccharides. These active components are believed to have potential effects such as promoting cell proliferation, regulating inflammatory responses, and improving tissue repair, making them suitable for the degenerative repair of lumbar disc herniation and intervention for nerve root inflammation. However, existing topical deer antler preparations mostly employ simple pulverization, extraction, or ointment-based mixing methods, lacking graded extraction, stabilization and protection, and efficient delivery design for active components such as deer antler polypeptides and natural growth factors. Therefore, improving the transdermal delivery efficiency and local utilization rate of deer antler active components is a key issue in the development of topical deer antler preparations.

[0006] Microneedling technology offers a new technical approach to address the challenges of transdermal delivery of large molecule drugs. Microneedles can penetrate the stratum corneum in a minimally invasive manner, forming micron-sized drug delivery channels on the skin surface, thereby significantly improving the transdermal delivery efficiency of hydrophilic drugs, peptides, and proteins. Soluble microneedles can also gradually dissolve after insertion into the skin, releasing drugs directly to the epidermis or superficial dermis, combining convenient administration with high patient acceptability. However, existing microneedle formulations typically focus on rapid, single-dose administration, lacking sufficient depth and timing of drug release, making it difficult to simultaneously meet the needs of rapid onset of action during acute pain in lumbar disc herniation and continuous intervention during chronic degeneration.

[0007] Furthermore, drug release time is limited after microneedle administration alone, making it difficult to maintain effective local concentrations for extended periods. Liposomes, as nanodelivery systems composed of a phospholipid bilayer, can encapsulate hydrophilic or amphiphilic active ingredients and provide some protection for peptide drugs, thus improving drug stability and delivery efficiency. Combining liposome delivery systems with microneedle structures and further constructing a reservoir layer can achieve sustained-release delivery on top of rapid microneedle delivery, forming a drug delivery mode that combines rapid and sustained release. However, the structural integration and sequential delivery design of microneedle layers and liposome reservoir layers are still relatively rare in existing topical patches.

[0008] In the process of lumbar disc herniation and disc degeneration, local inflammatory responses and matrix degradation are often accompanied by abnormal expression of matrix metalloproteinases, among which MMP-2 and MMP-13 are correlated with extracellular matrix degradation, collagen destruction, and degenerative progression. If these pathologically related enzymes can be used as stimulating signals to design enzyme-responsive controlled-release carriers, linking the drug release rate to enzyme levels in the pathological microenvironment, it is hoped that the specificity and controllability of drug release can be improved. Existing topical formulations are mostly passive diffusion or conventional sustained-release formulations, which typically cannot respond to the characteristics of the pathological microenvironment, making it difficult to achieve more precise on-demand dosing.

[0009] Meanwhile, oxidative stress is also a significant contributing factor to lumbar intervertebral disc degeneration and inflammation. Excessive reactive oxygen species can exacerbate nucleus pulposus cell damage, promote the release of inflammatory factors and matrix degradation, further accelerating the degenerative process. Cerium oxide nanoparticles, due to their Ce... 3+ / Ce 4+ The reversible valence state transition characteristic suggests the potential to mimic the activity of antioxidant enzymes; polydopamine coating can improve the dispersion stability of nanoparticles and provide a certain synergistic antioxidant effect. Therefore, introducing CeO2@PDA nanozymes into an external controlled-release system is expected to help scavenge excessive reactive oxygen species locally and improve the inflammation- and oxidative stress-related microenvironment.

[0010] In summary, existing topical formulations for lumbar disc herniation still suffer from problems such as low transdermal efficiency of large molecular active ingredients, a single drug release mode, lack of sequential delivery design, inability to respond to the pathological microenvironment, and lack of antioxidant microenvironment regulation function. Therefore, it is necessary to develop a traditional Chinese medicine plaster and its preparation method that integrates soluble microneedle penetration enhancement, stable delivery of deer antler active components, liposome reservoir sustained release, MMP-2 / MMP-13 dual-enzyme responsive controlled release, and CeO2@PDA nanozyme antioxidant regulation, to meet the comprehensive needs of rapid analgesia, anti-inflammation, and continuous repair intervention for lumbar disc herniation. Summary of the Invention

[0011] To address the aforementioned deficiencies, this invention provides a traditional Chinese medicine plaster for lumbar disc herniation and its preparation method, aiming to rapidly deliver macromolecular deer antler active ingredients to the dermis for rapid onset of action, and to intelligently adjust the drug release rate according to the pathological state of the lesion for on-demand drug release.

[0012] The specific plan is as follows: The first aspect of this invention provides a method for preparing a traditional Chinese medicine plaster for lumbar disc herniation, comprising the following steps: S1, Pre-treatment of Chinese herbal raw materials: The Chinese herbal raw materials used include deer antler, drynaria, eucommia, angelica pubescens, chuanxiong, borneol, and menthol; S2, Graded extraction and processing of active components from deer antler: S2.1, Prepare VAP lyophilized powder; S2.2, Prepare freeze-dried powder of deer antler macromolecular active components; S2.3, prepare VAP nanoliposome suspension; S2.4, Preparation of borneol / menthol / β-cyclodextrin inclusion complex; S3, extracting and purifying other Chinese medicinal materials besides deer antler to prepare freeze-dried powder of Chinese medicinal compound extract; Preparation of S4, MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres: S4.1, Prepare MMP-2 responsive peptide crosslinking agent and MMP-13 responsive peptide crosslinking agent; S4.2 Preparation of core-shell structured dual-enzyme responsive microspheres: The microspheres consist of an outer shell layer and an inner core layer. The outer shell layer is prepared based on the MMP-2 responsive polypeptide cross-linking agent, and the inner core layer is prepared based on the MMP-13 responsive polypeptide cross-linking agent, VAP lyophilized powder, and lyophilized powder of deer antler macromolecular active components. The MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres are prepared by combining these components. S5, Preparation of CeO2@PDA nanozyme; S6, Fabrication of soluble hierarchical loaded microneedle array: S6.1, Prepare the microneedle mold; S6.2, Prepare the PVP / HA matrix solution; S6.3, Preparation of graded loading solutions: Drug loading solution in the needle tip region: Take the VAP nanoliposome suspension, add the borneol / menthol / β-cyclodextrin inclusion complex and PVP / HA matrix solution to obtain the drug loading matrix solution in the needle tip region; Drug solution in the middle of the needle body and drug solution at the base of the needle body: Take the freeze-dried powder of the Chinese herbal compound extract and add it to the PVP / HA matrix solution to obtain the drug-loaded matrix solution in the middle of the needle body and the drug-loaded matrix solution at the base of the needle body; S6.4, Preparation of microneedle array film: The drug-loaded matrix liquid in the needle tip area is filled into the needle tip part of the mold, the drug-loaded matrix liquid in the middle part of the needle body is filled into the middle part of the mold needle body, and the drug-loaded matrix liquid in the base part of the needle body is filled into the base part of the needle body. After drying, a microneedle array film is obtained. S7, Preparation of liposome transition reservoir layer: Prepare PVP / PVA solution, add the VAP nanoliposome suspension and the freeze-dried powder of the deer antler macromolecular active component to the PVP / PVA solution to obtain a mixture, and coat the mixture on the backing surface of the microneedle array membrane to form a liposome transition reservoir layer. S8, Preparation of enzyme-responsive hydrogel controlled-release layer: S8.1, Preparation of hydrogel prepolymer solution: Take PVA solution, sodium alginate solution, and GelMA solution, mix them, add photoinitiator, the freeze-dried powder of the Chinese herbal compound extract, the CeO2@PDA nanozyme and the MMP-2 / MMP-13 dual enzyme cascade response microspheres to prepare hydrogel prepolymer solution. S8.2, the hydrogel prepolymer solution is poured onto the microneedle array membrane, and after treatment, an enzyme-responsive hydrogel controlled-release layer is formed.

[0013] Preferably, the following Chinese medicinal materials are used in S1: 30 parts deer antler, 15 parts drynaria rhizome, 12 parts eucommia bark, 10 parts angelica pubescens root, 8 parts chuanxiong rhizome, 3 parts borneol, and 2 parts menthol.

[0014] Preferably, the specific steps for preparing VAP lyophilized powder in step S2.1 are as follows: Fresh deer antler was freeze-dried and then pulverized to obtain freeze-dried deer antler powder. The freeze-dried deer antler powder was added to pre-cooled PBS buffer, and then papain was added for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the temperature was raised to inactivate the papain. After centrifugation, the supernatant was collected. The supernatant was sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa for fractionation to obtain VAP fractions with molecular weight ranges of 3–10 kDa. The retentate from the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain VAP freeze-dried powder. The specific steps for preparing the freeze-dried powder of deer antler macromolecular active components in step S2.2 are as follows: The retentate from the 10 kDa ultrafiltration membrane in S2.1 was collected and freeze-dried to obtain freeze-dried powder of deer antler macromolecular active components.

[0015] Preferably, the specific steps for preparing the VAP nanoliposome suspension in step S2.3 are as follows: Take egg yolk lecithin and cholesterol and place them in a container. Add anhydrous ethanol and dissolve them completely. Then, evaporate them under vacuum until a uniform and transparent lipid film is formed on the container wall. Take the VAP lyophilized powder, dissolve it in PBS buffer, and prepare a VAP solution; VAP solution is added to the container, glass beads are added, and the mixture is rotated to hydrate and detach the lipid membrane, forming a liposome suspension. The liposome suspension was subjected to probe sonication under ice bath conditions, and then granulated through a filter membrane to obtain VAP nanoliposome suspension.

[0016] Preferably, the specific steps for preparing the MMP-2 responsive peptide crosslinking agent and the MMP-13 responsive peptide crosslinking agent in step S4.1 are as follows: First, MMP-2 responsive peptide precursors and MMP-13 responsive peptide precursors with amino linkages at both ends were prepared by solid-phase peptide synthesis. The recognition sequence of the MMP-2 responsive peptide is GPLGIAGQ, and the recognition sequence of the MMP-13 responsive peptide is PQGLA. Then, N-acryloyloxysuccinimide ester was used to acryloylate the amino groups at both ends of the MMP-2 responsive peptide precursor and the MMP-13 responsive peptide precursor, respectively, to obtain the MMP-2 responsive peptide crosslinking agent Acryloyl-GPLGIAGQ-Acryloyl and the MMP-13 responsive peptide crosslinking agent Acryloyl-PQGLA-Acryloyl.

[0017] Preferably, the specific steps for preparing the core-shell dual-enzyme responsive microspheres in step S4.2 are as follows: Take polyethylene glycol diacrylate, add Acryloyl-GPLGIAGQ-Acryloyl and a photoinitiator to form a shell prepolymer liquid; A glass capillary microfluidic device was used to form W / O / W double droplets with polyvinyl alcohol aqueous solution as the inner phase, shell prepolymer liquid as the outer phase, and mineral oil containing Tween 20 as the collecting phase. The shell was photocrosslinked and cured by ultraviolet light irradiation to obtain hollow microspheres. Sodium alginate was added to Acryloyl-PQGLA-Acryloyl, and after dissolution, the VAP lyophilized powder and the deer antler macromolecular active component lyophilized powder were added to serve as the core prepolymer solution. Hollow microspheres were immersed in a core prepolymer solution to allow the solution to fully penetrate the hollow cavity of the microspheres. The microspheres were then removed and immersed in a calcium chloride solution for cross-linking and curing at room temperature. Finally, they were photocross-linked with ultraviolet light to obtain MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres.

[0018] Preferably, the specific steps for preparing CeO2@PDA nanozymes in step S5 are as follows: Cerium nitrate hexahydrate is dissolved in water, and ammonia is added to adjust the pH to alkaline, causing cerium ions to precipitate and yielding the cerium hydroxide precursor. Cerium hydroxide precursor was calcined and then cooled to room temperature to obtain cerium oxide nanoparticles; Cerium oxide nanoparticles were dispersed in Tris-HCl buffer, dopamine hydrochloride was added, the precipitate was collected by centrifugation, and the CeO2@PDA nanozyme was obtained by freeze-drying.

[0019] Preferably, the specific operation of S8.2 is as follows: The microneedle array membrane is laid flat at the bottom of the mold with the microneedle tips facing down. The hydrogel prepolymer obtained in S8.1 is poured onto the microneedle array membrane. The mold is then frozen, removed, and thawed at room temperature. The freeze-thaw cycle is repeated multiple times to form a PVA physical crosslinking network. After the freeze-thaw cycle is completed, the mold is placed under a UV lamp to irradiate it, causing the GelMA to undergo photocrosslinking and form a chemical crosslinking network, and finally the mold is demolded.

[0020] The second aspect of the present invention provides a traditional Chinese medicine plaster for lumbar disc herniation, which is prepared by the method for preparing traditional Chinese medicine plaster for lumbar disc herniation as described in the first aspect of the present invention.

[0021] Preferably, the front of the plaster has a medical breathable non-woven fabric backing layer, and the back of the plaster has a release protective layer.

[0022] The beneficial effects of this invention are as follows: (1) This invention successfully solves the industry problem of large molecular weight, strong hydrophilicity, and difficulty in penetrating the stratum corneum of deer antler polypeptides through a triple technology of enzymatic hydrolysis, phospholipid complex modification, and physical permeation enhancement using microneedle arrays. This effect allows deer antler, a precious medicinal material, to act directly on dermal fibroblasts and immune cells, avoiding the defects of low oral bioavailability and gastrointestinal irritation, and expanding the new application of deer antler in the treatment of lumbar disc herniation.

[0023] (2) This invention introduces MMP-2 / MMP-13 dual-enzyme cascade-responsive microspheres into an enzyme-responsive hydrogel controlled-release layer. Utilizing the abnormal expression of matrix metalloproteinases in the pathological processes related to lumbar disc herniation and disc degeneration, it achieves a correlation regulation between drug release and the pathological microenvironment. The outer shell of the microspheres responds to MMP-2 degradation, while the inner core responds to MMP-13 to further release antler polypeptides and macromolecular active components of antler, thus forming a cascade-triggered release mechanism from the outside in. This design can increase the drug release rate when the pathological enzyme level is high and maintain a relatively sustained-release state when the enzyme level is low, which is beneficial for reducing unnecessary release and improving drug utilization efficiency.

[0024] (3) This invention utilizes a three-layer synergistic design to rapidly pierce the stratum corneum and release deer antler polypeptides and analgesic and anti-inflammatory components through a soluble microneedle layer, achieving rapid onset of action; utilizes a liposome transition reservoir layer to protect and provide secondary sustained release of the active ingredients of deer antler, maintaining local drug concentration; and utilizes an enzyme-responsive hydrogel controlled-release layer loaded with dual-enzyme-responsive microspheres and CeO2@PDA nanozymes to achieve continuous controlled release and regulation of the oxidative stress microenvironment, thereby addressing both acute analgesia and anti-inflammation and chronic repair intervention needs. Attached Figure Description

[0025] Figure 1 This is a scanning electron microscope (SEM) image of the herbal plaster of the present invention; Figure 2 This is a transmission electron microscope (TEM) image of the MMP-2 / MMP-13 dual-enzyme cascade-responsive microspheres in the herbal plaster of this invention; Figure 3 This is a transmission electron microscope (TEM) image of CeO2@PDA nanozyme in the traditional Chinese medicine plaster of this invention; Figure 4 This is a graph showing the effect of the amount of MMP-2 / MMP-13 dual-enzyme cascade-responsive microspheres added to the herbal plaster of this invention on the drug release performance. Figure 5 This is a drug release curve of the MMP-2 / MMP-13 cascade response of the traditional Chinese medicine plaster of this invention; Figure 6 This is a comparative diagram of the in vitro transdermal performance of the herbal plaster of this invention; Figure 7This is a graph showing the ROS scavenging activity of the CeO2@PDA nanozyme in the traditional Chinese medicine plaster of this invention. Detailed Implementation

[0026] The technical solutions of the present invention will now be clearly and completely described with reference to the accompanying drawings in the embodiments of the present invention.

[0027] Example 1: This embodiment provides a method for preparing a traditional Chinese medicine plaster for lumbar disc herniation, including the following steps: S1, Pretreatment of Chinese herbal raw materials: This embodiment uses the following parts by weight of traditional Chinese medicine raw materials: 30 parts deer antler, 15 parts *Drynaria fortunei*, 12 parts *Eucommia ulmoides*, 10 parts *Angelica pubescens*, 8 parts *Ligusticum chuanxiong*, 3 parts borneol, and 2 parts menthol. Deer antler refers to the unossified, densely hairy young antler of the male sika deer (Cervidae family). *Drynaria fortunei* is the dried rhizome of *Drynaria fortunei* (Polypodiaceae family), *Eucommia ulmoides* is the dried bark of *Eucommia ulmoides* (Eucommia family), *Angelica pubescens* is the dried root of *Angelica sinensis* (Apiaceae family), *Ligusticum chuanxiong* is the dried rhizome of *Ligusticum chuanxiong* (Apiaceae family), borneol is synthetic borneol (purity ≥98%), and menthol is a monoterpene compound extracted from peppermint oil (purity ≥99%). All traditional Chinese medicine raw materials were pulverized and passed through a 60-mesh sieve before use.

[0028] S2, Graded extraction and processing of active components from deer antler: S2.1, Preparation of VAP (Deer Antler Polypeptide) Lyophilized Powder: Take 30 parts by weight of fresh deer antler, remove the surface hair, cut into thin slices with a thickness of 2-3 mm, pre-freeze in an ultra-low temperature freezer at -80℃ for 12 hours, then transfer to a freeze dryer and freeze-dry at -50℃ and a vacuum degree <0.1 mbar for 48 hours. Grind the freeze-dried deer antler slices using a high-speed grinder and pass through a 100-mesh sieve to obtain freeze-dried deer antler powder. Further weigh 20 g of freeze-dried deer antler powder and add 300 mL of phosphate-buffered saline (PBS, 0.01 M, pH 7.4) pre-cooled to 4℃ at a material-to-liquid ratio of 1:15 (w / v). After stirring evenly, add 0.5 g of papain to obtain a mixed solution. Then place the mixed solution in an ultrasonic cell disruptor and perform ultrasonic-assisted enzymatic hydrolysis under ice bath conditions. The ultrasonic power is set to 200 W, using pulse mode (10 seconds of operation, 5 seconds of interval), with a total ultrasonic time of 20 minutes. Throughout the process, the temperature of the mixed solution is maintained below 10℃ using an ice bath. After ultrasonic treatment, the mixed solution was transferred to a constant-temperature shaking water bath and enzymatically hydrolyzed for 4 hours at 37°C and 100 rpm. After hydrolysis, the mixed solution was rapidly heated to 90°C and held for 10 minutes to completely inactivate papain. After cooling to room temperature, it was centrifuged at 10,000 rpm for 15 minutes at 4°C, and the supernatant was collected. The supernatant was further fractionated by sequentially passing it through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa. First, the solution was treated with a 10 kDa ultrafiltration membrane, and the permeate (<10 kDa fraction) was collected. Then, this permeate was treated with a 3 kDa ultrafiltration membrane, and the retentate was collected, yielding VAP (deer antler polypeptide) fractions with a molecular weight range of 3–10 kDa. The collected retentate was freeze-dried at -50°C for 48 hours to obtain VAP lyophilized powder.

[0029] S2.2, Preparation of freeze-dried powder of deer antler macromolecular active components: The retentate obtained after treatment with a 10 kDa ultrafiltration membrane in S2.1 was collected as the deer antler macromolecular active component with a molecular weight greater than 10 kDa. This component mainly includes macromolecular proteins, peptides, and other water-soluble macromolecular active substances derived from deer antler. The retentate was placed in a freeze dryer and freeze-dried at -50°C and a vacuum degree <0.1 mbar for 48 hours to obtain freeze-dried powder of the deer antler macromolecular active component, which was then sealed and stored at -20°C for later use.

[0030] S2.3, Preparation of VAP nanoliposome suspension: VAP nanoliposomes were prepared using a thin-film dispersion-ultrasound method. 800 mg of egg yolk lecithin and 200 mg of cholesterol were accurately weighed and placed in a 250 mL round-bottom flask. 20 mL of anhydrous ethanol was added, and the mixture was sonicated for 5 minutes to ensure complete dissolution. The round-bottom flask was then connected to a rotary evaporator and rotary evaporated for 40 minutes at a water bath of 45°C, a rotation speed of 80 rpm, and a vacuum of -0.08 MPa, until a uniform and transparent lipid film formed on the flask wall. The round-bottom flask was removed, and residual ethanol was removed by purging with nitrogen for 5 minutes. Further, 100 mg of the VAP lyophilized powder prepared in step S2.1 was dissolved in 40 mL of phosphate buffer (pH 6.8, 0.01 M) to prepare a VAP solution with a concentration of 2.5 mg / mL. The VAP solution was added to the aforementioned flask, along with an appropriate amount of 0.5 mm diameter glass beads. The mixture was then hydrated in a 45°C water bath at 150 rpm for 30 minutes, allowing the lipid film to fully hydrate and detach, forming a milky white liposome suspension. The liposome suspension was further transferred to a 50 mL centrifuge tube and placed in an ultrasonic cell disruptor. Ultrasonic treatment was performed under ice bath conditions with the following parameters: power 100 W, pulse mode (5 seconds on, 3 seconds off), and total sonication time 5 minutes. The sonicated liposome suspension was then granulated through 0.45 μm and 0.22 μm microporous membranes to obtain a VAP nanoliposome suspension.

[0031] S2.4, Preparation of borneol / menthol / β-cyclodextrin inclusion complex: Take 10 g of β-cyclodextrin, add 80 mL of distilled water, heat to 60℃ and stir to dissolve, preparing a saturated aqueous solution of β-cyclodextrin. Separately, take 1.5 g of borneol and 1.0 g of menthol, add 5 mL of anhydrous ethanol and dissolve. Under constant temperature and magnetic stirring at 60℃, slowly add the borneol / menthol ethanol solution dropwise to the saturated aqueous solution of β-cyclodextrin at a rate of about 1 mL / min. After the addition is complete, continue stirring at 60℃ for 2 hours to encapsulate the inclusion complex. After cooling the inclusion complex to room temperature, refrigerate it overnight at 4℃, filter, wash the precipitate twice with a small amount of anhydrous ethanol, dry it in a vacuum drying oven at 40℃ for 12 hours, grind it through an 80-mesh sieve, and obtain 4.85 g of borneol / menthol / β-cyclodextrin inclusion complex.

[0032] S3 involves extracting and purifying other Chinese medicinal materials besides deer antler to prepare freeze-dried powder of compound Chinese medicine extract.

[0033] Take 15 parts by weight of Drynaria fortunei, 12 parts by weight of Eucommia ulmoides, 10 parts by weight of Angelica pubescens, and 8 parts by weight of Ligusticum chuanxiong. Mix them, pulverize them through a 40-mesh sieve, place them in a round-bottom flask, add 70% (v / v) ethanol solution (solid to liquid ratio 1:10, w / v), heat and reflux for 2 hours, filter and collect the extract. Add 8 times the amount of 70% (v / v) ethanol to the filter residue again, reflux for 1.5 hours in the same way, and filter. Combine the two extracts and concentrate under reduced pressure at 50℃ until there is no alcohol odor, to obtain about 200 mL of concentrate. Pass the concentrate through a pretreated D101 macroporous adsorption resin column (column volume 500 mL) at a flow rate of 2 BV / h. After loading, first elute with 5 BV of distilled water to remove water-soluble impurities, then elute with 5 BV of 60% (v / v) ethanol solution at a flow rate of 3 BV / h, and collect the 60% ethanol eluent. The eluent was concentrated under reduced pressure at 50°C to a thick paste, then transferred to a freeze dryer and dried at -50°C and <0.1 mbar for 48 hours to obtain 23.5 g of freeze-dried powder of the traditional Chinese medicine compound extract.

[0034] Preparation of S4, MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres: S4.1, Preparation of MMP-2 (gelatinase A) responsive peptide cross-linking agents and MMP-13 (collagenase 3) responsive peptide cross-linking agents: To enable MMP-responsive peptides to participate in the subsequent photocrosslinking reaction of microspheres as crosslinking agents, MMP-responsive peptide precursors with amino-terminal linkers were first prepared using a solid-phase peptide synthesis method. Then, the amino groups at both ends of the peptide precursors were acrylated using N-acryloyloxysuccinimide ester to obtain a bi-acrylated MMP-responsive peptide crosslinking agent. Specifically, the recognition sequence of the MMP-2 responsive peptide is Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, i.e., GPLGIAGQ; and the recognition sequence of the MMP-13 responsive peptide is Pro-Gln-Gly-Leu-Ala, i.e., PQGLA. To facilitate subsequent bi-acrylation modification, amino-terminated flexible linkers were introduced at both ends of the above peptide sequences during solid-phase synthesis, resulting in the following peptide precursors: the MMP-2 responsive peptide precursor is: H2N-PEG-GPLGIAGQ-PEG-NH2. The MMP-13 responsive peptide precursor is: H2N-PEG-PQGLA-PEG-NH2, where PEG represents a short-chain hydrophilic polyethylene glycol linker. The linker improves the solubility of the peptide cross-linking agent in the aqueous system and reduces steric hindrance near the subsequent cleavage sites, making it easier for MMP-2 or MMP-13 to recognize the corresponding cleavage sequences.

[0035] (1) Preparation of MMP-2 responsive peptide precursor; The MMP-2 responsive peptide precursor H2N-PEG-GPLGIAGQ-PEG-NH2 was prepared using the Fmoc solid-phase peptide synthesis method. The specific preparation method was as follows: 0.5 g of Fmoc-Rink Amide resin was weighed and washed three times with 3 mL of N,N-dimethylformamide (DMF) each time to remove impurities. Further deprotection was performed with 20% piperidine / DMF solution for 10 minutes, repeated once, followed by five washes with DMF to obtain the resin.

[0036] Fmoc-amino acids were sequentially coupled, including: Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, and Fmoc-Gln(Trt)-OH. For each Fmoc-amino acid, the following steps were repeated: A coupling solution was prepared by dissolving 3 molar amounts of the Fmoc-amino acid, 3 molar amounts of N,N'-diisopropylcarbodiimide (DIC), and 3 molar amounts of 1-hydroxybenzotriazole (HOBt) in N-methylpyrrolidone (NMP). The coupling solution was added to the resin and stirred at room temperature for 1–2 hours. After coupling, the resin was washed 5 times with DMF. Fmoc was further removed with 20% piperidine / DMF, repeated once, and the resin was washed 5 times with DMF.

[0037] After polypeptide sequence assembly, amino-modified PEG linkers (H2N-PEG-NH2) were coupled at the N-terminus and C-terminus, respectively: 2–3 molar amounts of PEG and 2–3 molar amounts of DIC / HOBt were reacted in NMP for 2 hours to ensure complete coupling. The polypeptide was washed 5 times with DMF. Lysis was performed for 2–3 hours using a mixture of trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / deionized water = 95:2.5:2.5 (v / v / v) with gentle stirring at room temperature. The polypeptide was precipitated with cold diethyl ether, and the precipitate was collected by centrifugation and washed 2–3 times. The precipitate was dissolved in a deionized water mixture, and insoluble matter was removed by filtration. Preparative reversed-phase HPLC purification was performed: mobile phase A: 0.1% TFA aqueous solution, mobile phase B: 0.1% TFA acetonitrile solution, with gradient elution. The target peak fraction was collected, acetonitrile was removed by rotary evaporation, and the final product H2N-PEG-GPLGIAGQ-PEG-NH2 was obtained by freeze-drying and stored at -20℃ protected from light for later use.

[0038] (2) Preparation of MMP-13 responsive peptide precursor; The MMP-13 responsive peptide precursor H2N-PEG-PQGLA-PEG-NH2 was prepared using the same Fmoc solid-phase peptide synthesis method as described above. Using 0.5 g of Fmoc-Rink Amide resin as the solid-phase support, amino acids were sequentially coupled according to the amino acid sequence of PQGLA, and aminolated PEG linkers were introduced at both ends of the peptide sequence. After the peptide chain was assembled, it was cleaved by trifluoroacetic acid, precipitated with cold diethyl ether, washed by centrifugation, purified by preparative reversed-phase high-performance liquid chromatography, and freeze-dried to obtain the MMP-13 responsive peptide precursor H2N-PEG-PQGLA-PEG-NH2.

[0039] (3) Preparation of MMP-2 responsive peptide crosslinking agent with dual-terminal acrylylation; Weigh 200 mg of the MMP-2 responsive peptide precursor H2N-PEG-GPLGIAGQ-PEG-NH2 and add it to 10 mL of sodium bicarbonate buffer (pH 8.0–8.5). Stir until fully dissolved to prepare an MMP-2 responsive peptide precursor solution. Separately, dissolve N-acryloyloxysuccinimide ester in a small amount of anhydrous dimethyl sulfoxide or anhydrous N,N-dimethylformamide to prepare an acrylation reagent solution.

[0040] Under ice bath and light-protected conditions, the acrylamide reagent solution was slowly added dropwise to the MMP-2 responsive peptide precursor solution. The preferred molar ratio of N-acryloyloxysuccinimide ester to the amino groups in the peptide precursor was 2.2–4.0:1 to ensure sufficient reaction of the amino groups at both ends of the peptide. After the addition was complete, the reaction was stirred at 0–5°C for 1–2 hours, then allowed to proceed to room temperature and light protection for 8–12 hours, allowing the amino groups at both ends of the peptide precursor to undergo an amidation reaction with the N-acryloyloxysuccinimide ester, introducing acryloyl groups.

[0041] After the reaction was complete, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 500 Da or 1000 Da. Deionized water was used as the dialysis medium, and the solution was dialyzed at 4°C for 24–48 hours, with the dialysate changed every 4–6 hours to remove unreacted acrylamide, N-hydroxysuccinimide, organic solvents, and inorganic salts. After dialysis, the dialysate was freeze-dried to obtain the crude product.

[0042] The crude product was further purified by preparative reversed-phase high-performance liquid chromatography (RP-HPLC). Mobile phase A consisted of water containing 0.1% trifluoroacetic acid, and mobile phase B consisted of acetonitrile containing 0.1% trifluoroacetic acid. Gradient elution was used, and the target peak fraction was collected. After removing the acetonitrile, the target fraction was freeze-dried to obtain the acryloyl-terminated MMP-2 responsive peptide crosslinking agent: Acryloyl-PEG-GPLGIAGQ-PEG-Acryloyl. It was stored at -20°C in a light-protected, sealed container for later use.

[0043] (4) Preparation of MMP-13 responsive peptide crosslinking agent with dual-terminal acrylylation; Weigh 200 mg of the MMP-13 responsive peptide precursor H2N-PEG-PQGLA-PEG-NH2 and add it to 10 mL of sodium bicarbonate buffer (pH 8.0–8.5). Stir to dissolve and prepare an MMP-13 responsive peptide precursor solution. Separately, dissolve N-acryloyloxysuccinimide ester in a small amount of anhydrous dimethyl sulfoxide or anhydrous N,N-dimethylformamide to prepare an acrylation reagent solution.

[0044] Under ice bath and light-protected conditions, the acrylamide reagent solution was slowly added dropwise to the MMP-13 responsive peptide precursor solution. The preferred molar ratio of the N-acryloyloxysuccinimide ester to the amino groups in the peptide precursor was 2.2–4.0:1. After the addition was complete, the reaction was continued at 0–5°C with stirring for 1–2 hours, followed by a reaction at room temperature in the dark for 8–12 hours to ensure complete acrylamide acrylation of the amino groups at both ends of the peptide precursor.

[0045] After the reaction, the reaction solution was transferred to a dialysis bag with a molecular weight cutoff of 500 Da or 1000 Da, and dialyzed with deionized water at 4°C for 24–48 hours to remove unreacted small molecules and solvent residues. After dialysis, the dialysate was freeze-dried to obtain the crude product. The crude product was purified by preparative reversed-phase high-performance liquid chromatography, the target peak was collected, organic solvents were removed, and the product was freeze-dried to obtain the acryloyl-terminated MMP-13 responsive peptide crosslinking agent: Acryloyl-PEG-PQGLA-PEG-Acryloyl. It was stored at -20°C in a light-protected, sealed container for later use.

[0046] (5) Reaction principle; In the above preparation process, the MMP-responsive peptide precursor contains primary amino groups at both ends. The active ester group in N-acryloyloxysuccinimide ester can undergo amidation with the primary amino groups, thereby introducing acryloyl groups at both ends of the peptide. The resulting double-terminated acryloylated peptide crosslinking agent simultaneously possesses two unsaturated double bonds that can participate in free radical photopolymerization and an MMP-recognizable enzyme cleavage sequence.

[0047] In the subsequent microsphere preparation process, Acryloyl-PEG-GPLGIAGQ-PEG-Acryloyl, as an MMP-2 responsive outer shell crosslinking agent, participated in the construction of the outer shell photocrosslinking network; Acryloyl-PEG-PQGLA-PEG-Acryloyl, as an MMP-13 responsive core layer crosslinking agent, participated in the construction of the core layer photocrosslinking network. When the corresponding matrix metalloproteinases are present in the system, the enzymatic sequences in the peptide crosslinking agents are recognized and cleaved, causing the crosslinking network to degrade, thereby promoting the release of the encapsulated drug and achieving MMP-2 / MMP-13 dual-enzyme cascade response controlled release.

[0048] S4.2, Preparation of core-shell dual-enzyme responsive microspheres: Preparation of the outer shell (MMP-2 responsive layer): Hollow microsphere shells were prepared using microfluidic technology. Specifically, polyethylene glycol diacrylate (PEGDA, weight-average molecular weight M...) was used... w =700 Da) 2.0 g was dissolved in 10 mL of deionized water, and 200 mg of Acryloyl-GPLGIAGQ-Acryloyl obtained in S4.1 and 50 mg of photoinitiator phenyl-2,4,6-trimethylbenzoyl lithium phosphite (LAP) were added. The mixture was stirred and dissolved to prepare the shell prepolymer solution. A glass capillary microfluidic device was used, and the inner phase fluid was 5% (w / v) polyvinyl alcohol (PVA, M w The microspheres consisted of an aqueous solution with a concentration of 13000–23000 Da, an outer phase prepolymerized liquid, and a collection phase containing 2% Tween 20 mineral oil. The flow rates were adjusted to 2 μL / min for the inner phase, 20 μL / min for the outer phase, and 200 μL / min for the collection phase, forming W / O / W dual droplets at the junction of the microfluidic chip. These droplets were then irradiated with 365 nm UV light (20 mW / cm², 30 s), initiating photocrosslinking and curing of the outer shell layer. The microspheres were collected, washed three times with isopropanol to remove the mineral oil, and then washed three times with deionized water to obtain hollow microspheres.

[0049] Filling and crosslinking of the core layer (MMP-13 response layer): 100 mg of Acryloyl-PQGLA-Acryloyl was dissolved in 10 mL of deionized water. 1.0 g of sodium alginate was added and stirred until dissolved. 50 mg of VAP lyophilized powder prepared in S2.1 and 30 mg of deer antler macromolecular active component lyophilized powder prepared in S2.2 were added and mixed thoroughly to form the core prepolymer solution. The hollow microspheres prepared above were immersed in the core prepolymer solution and kept in a vacuum drying oven (-0.08 MPa) for 30 minutes to allow the core prepolymer solution to fully penetrate the hollow cavity of the microspheres. The microspheres were then removed and quickly immersed in a 5% (w / v) calcium chloride (CaCl2) solution for crosslinking and curing at room temperature for 20 minutes to allow the sodium alginate and CaCl2 to crosslink and cure.2+ A calcium alginate gel core was formed. Simultaneously, the microspheres were irradiated with 365 nm ultraviolet light (20 mW / cm²) for 2 minutes to further photocrosslink Acryloyl-PQGLA-Acryloyl. The resulting microspheres were washed three times with deionized water to obtain core-shell structured MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres, such as... Figure 2 As shown.

[0050] S5, Preparation of CeO2@PDA nanozyme (cerium oxide nanozyme encapsulated in polydopamine): 4.34 g of cerium nitrate hexahydrate (Ce(NO3)3·6H2O) was dissolved in 100 mL of deionized water. 30% ammonia solution was slowly added dropwise under magnetic stirring until the solution pH reached 10, producing a pale yellow precipitate. The precipitate was separated by centrifugation (8000 rpm, 10 min), washed three times each with deionized water and anhydrous ethanol, and dried under vacuum at 60 °C for 12 h to obtain cerium hydroxide precursor. The cerium hydroxide precursor was placed in a muffle furnace and calcined at 400 °C at a rate of 5 °C / min for 4 h, then naturally cooled to room temperature to obtain cerium oxide nanoparticles. 200 mg of cerium oxide nanoparticles were ultrasonically dispersed in 100 mL of Tris-HCl buffer (10 mM, pH 8.5), and 100 mg of dopamine hydrochloride was added. The reaction was carried out under magnetic stirring at room temperature in the dark for 6 h. The reaction solution was dark brown. The precipitate was collected by centrifugation (12000 rpm, 15 minutes), washed three times with deionized water, and freeze-dried to obtain CeO2@PDA nanozyme. Figure 3 As shown.

[0051] S6, Fabrication of soluble hierarchical loaded microneedle array: S6.1, Preparation of microneedle mold: Microneedle negative molds were prepared using a polydimethylsiloxane (PDMS) replication molding method. Using a laser-engraved metal positive mold (microneedle height 550 μm, cone base diameter 300 μm, needle spacing 500 μm) as a template, PDMS prepolymer and curing agent were mixed uniformly at a mass ratio of 10:1, degassed under vacuum, and poured onto the surface of the positive mold. The mixture was cured at 70℃ for 4 hours, cooled, and then demolded to obtain the PDMS microneedle negative mold. Before use, the negative mold was ultrasonically cleaned with 75% ethanol for 15 minutes, rinsed with deionized water, and dried with nitrogen.

[0052] S6.2, Preparation of PVP / HA matrix solution: Weigh out 6.0 g of polyvinylpyrrolidone (PVP K30) and sodium hyaluronate (HA, M). w =10 kDa) 2.0 g, add 12 mL of deionized water, stir and dissolve at room temperature to obtain PVP / HA matrix solution.

[0053] S6.3, Preparation of graded loading solutions: Drug loading solution in the needle tip area: Take 5 mL of VAP nanoliposome suspension prepared in S2.3 (equivalent to about 10 mg of VAP), add 500 mg of borneol / menthol / β-cyclodextrin inclusion complex prepared in S2.4, and then add 2 mL of PVP / HA matrix solution prepared in S6.2. Stir magnetically until homogeneous to obtain drug loading matrix solution in the needle tip area (solution A). Drug solution in the middle of the needle body: Take 200 mg of the lyophilized powder of the compound extract of traditional Chinese medicine obtained in S3, dissolve it in 2 mL of deionized water, add 3 mL of the PVP / HA matrix solution prepared in S6.2, mix well, and obtain the drug-loaded matrix solution in the middle of the needle body (solution B). Drug solution at the base of the needle: Take 300 mg of the lyophilized powder of the compound extract of traditional Chinese medicine obtained in S3, dissolve it in 2 mL of deionized water, add 3 mL of the PVP / HA matrix solution prepared in S6.2, mix well, and obtain the drug-loaded matrix solution at the base of the needle (solution C).

[0054] S6.4, Fabrication of microneedle array membrane: A multi-stage centrifugation-layer filling process was employed. First, the S6.1 PDMS microneedle negative mold was placed in a 50 mL centrifuge tube adapter, with the mold needle tip facing down. First centrifugation filling (needle tip area): 200 μL of solution A was pipetted and evenly spread onto the mold surface. The centrifuge tube was placed in a low-temperature centrifuge and centrifuged at 4°C and 3000 rpm for 10 minutes to ensure that solution A fully filled the needle tip area. The mold was then removed, and excess solution was gently scraped off the mold surface with a soft scraper.

[0055] Second centrifugation filling (middle of the needle): Take 250 μL of solution B and spread it evenly on the surface of the mold. Centrifuge at 4℃ and 3000 rpm for 5 minutes to fill the middle of the needle body with solution B (approximately 150–350 μm in height). Remove the mold and scrape off any excess solution from the surface.

[0056] Third centrifugation filling (needle base): Take 300 μL of solution C and spread it evenly on the mold surface. Centrifuge at 4℃ and 3000 rpm for 5 minutes to fill the base of the needle with solution C (approximately 350–550 μm in height). Remove the mold and scrape off any excess solution from the surface.

[0057] The mold was placed in a desiccator and slowly dried for 24 hours at room temperature (25±2℃) and relative humidity of 30%. After drying, the microneedle array was carefully peeled off from the PDMS mold with pointed tweezers to obtain a soluble hierarchical loaded microneedle array film.

[0058] S7, Preparation of the liposome transition reservoir layer: Weigh out 1.5 g of PVP and polyvinyl alcohol (PVA, M).w =89000~98000 Da) 0.5 g, add 8 mL of deionized water, stir and dissolve in an 80℃ water bath, cool to room temperature to obtain a PVP / PVA solution. Take 3 mL of the VAP nanoliposome suspension prepared in S2.3 and 100 mg of the lyophilized powder of deer antler macromolecular active component prepared in S2.2 (pre-dissolved in 1 mL of deionized water), add to the above PVP / PVA solution to obtain a mixture, magnetically stir the mixture until homogeneous, and degas under vacuum for 30 minutes. Lay the microneedle array membrane prepared in S6.4 flat on a clean glass plate with the needle tips facing down, and use a coater to evenly coat the above mixture on the backing surface of the microneedle array, controlling the coating thickness to about 200 μm of wet film. Place the glass plate on a horizontal platform and allow it to dry naturally at room temperature for 6 hours to form a liposome transition reservoir layer with a thickness of 55~70 μm on the microneedle array membrane of S6.

[0059] S8, Preparation of enzyme-responsive hydrogel controlled-release layer: S8.1, Preparation of hydrogel prepolymer solution: Weigh 4.0 g of PVA, add 36 mL of deionized water, and stir to dissolve in a 90°C water bath for 2 hours. Cool to room temperature to prepare a 10% (w / v) PVA solution. Weigh 1.0 g of sodium alginate, add 49 mL of deionized water, and stir to dissolve overnight at room temperature to prepare a 2% (w / v) sodium alginate solution. Weigh 2.0 g of methacrylamide gelatin (GelMA), add 18 mL of deionized water, and stir to dissolve in a 50°C water bath to prepare a 10% (w / v) GelMA solution. Take 20 mL of the above PVA solution, 20 mL of sodium alginate solution, and 10 mL of GelMA solution, and mix thoroughly. Add 100 mg of photoinitiator LAP, 2.0 g of lyophilized powder of traditional Chinese medicine compound extract prepared in S3, 200 mg of CeO2@PDA nanozyme prepared in S5 (ultrasonically dispersed in 2 mL of deionized water), and 5.0 g of MMP-2 / MMP-13 dual enzyme cascade responsive microspheres prepared in S4.2 (wet weight, equivalent to about 1.2 g dry weight). Mix evenly with magnetic stirring and degas under vacuum for 30 minutes to obtain hydrogel prepolymer solution.

[0060] S8.2, Integrated assembly of the three-layer structure. The microneedle array membrane prepared in S7 was laid flat at the bottom of a mold (internal dimensions 5 cm × 5 cm, depth 2 mm), with the microneedle tips facing downwards. The hydrogel prepolymer obtained in S8.1 was slowly poured onto the membrane to a thickness of approximately 1.5 mm. The mold was frozen at -20°C for 2 hours, then thawed at room temperature for 2 hours. This freeze-thaw cycle was repeated 3 times to form a physical cross-linked PVA network. After the freeze-thaw cycle, the mold was placed under a 365 nm UV lamp (light intensity 20 mW / cm²) for 5 minutes to induce photocrosslinking of the GelMA, forming a chemical cross-linked network. Simultaneously, the sodium alginate in the hydrogel reacts with trace amounts of Ca in the skin exudate during subsequent use. 2+ Ionic cross-linking occurs, ultimately forming a triple interpenetrating network structure of PVA (physical cross-linking), calcium alginate (ionic cross-linking), and GelMA (chemical cross-linking). After demolding, an enzyme-responsive hydrogel controlled-release layer with a thickness of 1.05–1.15 mm is formed on the surface of the liposome transition reservoir layer.

[0061] After the above three layers are assembled, a medical-grade breathable non-woven fabric backing layer is integrated on the front of the patch, and a removable release protective layer is integrated on the back. Therefore, the overall structure of the patch from bottom to top is as follows: release protective layer (removed before use) — soluble microneedle layer — liposome transition reservoir layer — enzyme-responsive hydrogel controlled-release layer — medical-grade breathable non-woven fabric backing layer. A 3 mm wide medical pressure-sensitive adhesive ring is provided around the perimeter of the patch for application and fixation. Finally, a silicone release paper is placed on top, the patch is packaged in an aluminum foil bag, and sealed to obtain the herbal patch of this invention. Figure 1 As shown.

[0062] Example 2 (Optimization of the ratio of MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres in the enzyme-responsive hydrogel controlled-release layer): This example investigates the effect of the amount of dual-enzyme responsive microspheres added to the enzyme-responsive hydrogel controlled-release layer on drug release performance. Traditional Chinese medicine plasters were prepared according to the method in Example 1, except that the amount of MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres added in step S8.1 was changed. The amounts of MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres added were 2.5 g, 5.0 g, 7.5 g, and 10.0 g (wet weight), respectively, while other conditions remained unchanged. Four batches of plaster samples (numbered G1 to G4) were prepared.

[0063] The in vitro drug release behavior of each batch of plasters was evaluated using the Franz diffusion cell method. The receiving solution was PBS buffer (pH 7.4) containing 0.5% Tween 80. The experimental group's supply cell contained a mixed enzyme solution of MMP-2 (10 nM) and MMP-13 (10 nM) to simulate the pathological microenvironment, while the control group's supply cell contained no enzyme. Samples of 0.5 mL were taken at preset time points (1, 2, 4, 8, 12, 24, and 48 h), and an equal volume of isothermal receiving solution was added simultaneously. The VAP concentration in the samples was determined by HPLC (chromatographic conditions: C18 column, mobile phase: acetonitrile-0.1% trifluoroacetic acid aqueous solution gradient elution, detection wavelength: 214 nm).

[0064] The results are as follows Figure 4 As shown, with the increase of the amount of MMP-2 / MMP-13 dual-enzyme cascade-responsive microspheres, the cumulative drug release rate under enzyme conditions gradually increased. However, the enzyme response enhancement factor of G2 (5.0 g) had already reached 2.17 times, and further increasing the amount of microspheres had a slower effect on the increase in drug release rate. Moreover, excessive microsphere dosage may affect the mechanical properties of the hydrogel. Considering all factors, an addition amount of 5.0 g of MMP-2 / MMP-13 dual-enzyme cascade-responsive microspheres is the optimal choice.

[0065] Example 3 (MMP-2 / MMP-13 cascade response drug release validation): The MMP-2 / MMP-13 dual-enzyme cascade response microspheres prepared in Example 1 were placed in the following four release media for in vitro drug release experiments: medium A was PBS buffer (pH 7.4), medium B was PBS buffer containing MMP-2 (10 nM), medium C was PBS buffer containing MMP-13 (10 nM), and medium D was PBS buffer containing MMP-2 (10 nM) + MMP-13 (10 nM).

[0066] Microsphere samples and release media were mixed at a ratio of 1:100 (w / v) and placed in a 37°C constant-temperature shaking incubator (50 rpm). VAP concentrations were measured at set time points. Figure 5 As shown, the experimental results indicate that both single enzymes (MMP-2 or MMP-13) can promote drug release from microspheres, but the effect of MMP-2 is more significant (because it acts on the outer shell). When MMP-2 and MMP-13 are present simultaneously (medium D), the release rate is significantly accelerated and the cumulative release rate is the highest, reaching 79.3% after 24 hours, significantly higher than that of media B (52.7%) and media C (33.6%). This verifies the cascade triggering mechanism of MMP-2 degradation in the outer shell exposing the core, followed by MMP-13 degradation in the core layer accelerating drug release.

[0067] Example 4 (Comparative Experiment on In Vitro Transdermal Performance): Using a modified Franz diffusion cell method, and with isolated porcine abdominal skin (approximately 2.5 mm thick, with an intact stratum corneum) as the transdermal barrier, the transdermal performance of the following four groups of samples was compared: Experimental Group A (Complete Patch of Example 1): The microneedle layer was pressed into the skin and held for 60 seconds before application. Control Group B (Ordinary Hydrogel Patch without Microneedles): An enzyme-responsive hydrogel controlled-release layer was prepared according to the formulation of Example 1, without preparing the microneedle layer and liposome transition reservoir layer. Control Group C (Blank Hydrogel Matrix): Contained only PVA / sodium alginate / GelMA hydrogel, without drugs or MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres.

[0068] The receiving solution was PBS (pH 7.4) buffer containing 0.5% Tween 80, and the water bath temperature was 37±0.5℃ with magnetic stirring at 300 rpm. The drug delivery area of ​​the supply tank was 1.77 cm². Samples were taken at 2, 4, 8, 12, and 24 hours to determine the VAP concentration in the receiving solution and calculate the cumulative transdermal dose.

[0069] like Figure 6 As shown, the data indicates that experimental group A achieved a cumulative transdermal VAP dose of 312.5 μg / cm² within 24 hours, with a cumulative permeability of 68.4%, which is 3.5 times that of control group B (without microneedle hydrogel, 19.6%). Experimental group A reached a transdermal dose of 45.2 μg / cm² within 2 hours, demonstrating that microneedle puncture can rapidly establish transdermal channels and significantly shorten the drug's onset time.

[0070] Example 5 (Validation of ROS scavenging activity of CeO2@PDA nanozyme): The superoxide dismutase (SOD) simulated activity of CeO2@PDA nanozymes was determined using the pyrogallol autoxidation method. Tris-HCl buffer (50 mM, pH 8.2, containing 1 mM EDTA) was prepared, and different concentrations of CeO2@PDA nanozymes (0, 10, 20, 50, 100 μg / mL) were added. Pyrogallol solution (final concentration 0.2 mM) was then added, and the absorbance at 325 nm was immediately measured over time (recorded every 30 seconds for 5 minutes). The inhibition rate was calculated.

[0071] The results are as follows Figure 7 As shown, the CeO2@PDA nanozyme exhibited concentration-dependent SOD mimicry activity, with an inhibition rate of 72.3% at a concentration of 50 μg / mL. The CeO2@PDA nanozyme was co-incubated with H2O2 solution (1 mM), and the hydroxyl radical signal was detected using electron spin resonance (ESR). The results showed that 50 μg / mL CeO2@PDA nanozyme reduced the hydroxyl radical signal intensity by approximately 75%, confirming its significant ROS scavenging ability.

[0072] The principle of this invention is as follows: The herbal hydrogel patch for lumbar disc herniation prepared according to the method in Example 1 is based on the synergistic effect of the following five levels: active microneedle puncture to break through the stratum corneum barrier, liposome intermediate reservoir to achieve sequential drug delivery, MMP-2 / MMP-13 dual enzyme cascade response controlled release to achieve intelligent feedback of the pathological microenvironment, the cartilage repair and nerve regeneration mechanism of deer antler active components, and the reactive oxygen species scavenging and microenvironment regulation of cerium oxide nanoenzymes. The scientific principles are explained layer by layer below.

[0073] I. The synergistic permeation-enhancing principle of microneedle active puncture transdermal penetration and graded loading; The stratum corneum of the skin in the lumbar region is 15–20 μm thick, beneath which lies the erector spinae muscle group, which is 3–5 cm thick. Traditional Chinese medicine plasters rely on passive diffusion, requiring drug molecules to overcome the dense stratum corneum, resulting in insufficient transdermal penetration of large molecular active ingredients (such as deer antler polypeptide VAP, with a molecular weight of 3–10 kDa).

[0074] This invention uses a soluble microneedle array as the first transdermal layer, and its working principle includes: (1) Physical puncture to break through the stratum corneum barrier. The microneedle height is 500-550 μm, which is sufficient to puncture the epidermis and enter the superficial dermis. The microneedle body is composed of PVP and sodium hyaluronate, which has sufficient mechanical strength to penetrate the skin.

[0075] (2) Rapid dissolution to form a drug reservoir in the dermis. After needle insertion, the drug dissolves rapidly (within about 60 seconds), releasing the loaded drug directly into the active epidermis and superficial dermis. This mechanism bypasses the stratum corneum barrier and improves the bioavailability of macromolecular drugs such as VAP.

[0076] (3) Graded loading enables sequential treatment. The microneedle body is divided into three functional zones along the longitudinal axis: the tip zone (0–150 μm) is loaded with VAP nanoliposomes and borneol / menthol / β-cyclodextrin inclusion complexes, mainly for rapid dissolution and release after puncture, forming an initial high concentration of drug delivery; the middle zone (150–350 μm) is loaded with traditional Chinese medicine compound extracts, mainly for rapid release of analgesic and anti-inflammatory components; the base zone (350–550 μm) is further loaded with traditional Chinese medicine compound extracts and connects with the liposome transition reservoir layer to prolong the local drug delivery time. The above continuous zoning can avoid gaps in drug loading between the middle and base of the needle body, improving the continuity of the microneedle dissolution and drug release process.

[0077] II. The principle of secondary sustained release in the liposome transition reservoir layer; After the microneedles dissolve, the liposome transition reservoir layer of the patch adheres to the skin surface and performs the following functions: (1) Liposomes promote transcellular transport of VAP. VAP nanoliposomes are composed of egg yolk lecithin and cholesterol, which are highly similar to the keratinocyte membrane. Liposomes can directly deliver VAP to dermal fibroblasts and immune cells through cell membrane fusion and endocytosis, further improving the intracellular drug delivery efficiency. The liposome bilayer structure also protects VAP from degradation by skin enzymes.

[0078] (2) Continuous supply of macromolecular active components of deer antler. This layer is simultaneously loaded with macromolecular active components of deer antler with a molecular weight >10 kDa. These macromolecular active components are slowly released from the PVP / PVA film under the hydration of the skin surface, maintaining the local drug concentration for 6 to 8 hours and providing continuous growth factor support for deep tissue repair.

[0079] (3) Isolation and anti-burst release function. This layer is located between the microneedle layer and the enzyme-responsive hydrogel controlled release layer, and plays a physical isolation role to prevent the drug loaded in the enzyme-responsive hydrogel controlled release layer from non-specific burst release in the early stage of application, thus ensuring the controllability of drug release behavior.

[0080] III. The principle of drug release in the MMP-2 / MMP-13 dual-enzyme cascade response; The occurrence and development of lumbar disc herniation are accompanied by abnormally high expression of matrix metalloproteinases (MMPs) in the intervertebral disc microenvironment. Studies have shown that MMP expression during intervertebral disc degeneration exhibits clear temporal characteristics: Early stage (inflammation-dominated phase): Nucleus pulposus cells and infiltrating macrophages release large amounts of pro-inflammatory factors (TNF-α, IL-1β, etc.), inducing a significant upregulation of MMP-2 expression. MMP-2 (gelatinase A) mainly degrades basement membrane components and denatured collagen, participating in inflammatory cell infiltration and tissue edema.

[0081] Mid-to-late stage (matrix degradation stage): As inflammation persists, chondrocytes and nucleus pulposus cells secrete large amounts of MMP-13 (collagenase 3), which specifically degrades type II collagen and aggregated proteoglycans, leading to loss of intervertebral disc height and decreased mechanical properties.

[0082] Based on the above-mentioned pathological characteristics, this invention designs core-shell structured MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres, whose drug release mechanism is as follows: (1) MMP-2-specific degradation of the outer shell. The microsphere shell is composed of PEG hydrogel cross-linked with the MMP-2-responsive polypeptide sequence GPLGIAGQ. GPLGIAGQ is a specific recognition and enzymatic cleavage substrate sequence of MMP-2 (Gly-Pro-Leu-Gly↓Ile-Ala-Gly-Gln). When the patch is applied to the skin corresponding to the lesion of the lumbar vertebra, the active ingredients released by the microneedles and liposome transition reservoir are absorbed into the systemic circulation through the dermal capillary network and reach the local lesion of the intervertebral disc. The high concentration of MMP-2 in the lesion microenvironment recognizes the GPLGIAGQ sequence and undergoes specific enzymatic cleavage between Gly-Leu, resulting in the degradation of the PEG network in the outer shell, the rupture of the microsphere shell, and the exposure of the inner core layer.

[0083] (2) MMP-13-responsive accelerated drug release in the core layer. The core is composed of calcium alginate gel cross-linked with the MMP-13-responsive polypeptide sequence PQGLA. PQGLA (Pro-Gln-Gly↓Leu-Ala) is a specific recognition substrate of MMP-13. When the MMP-13 level in the lesion increases, the core layer is further enzymatically degraded, accelerating the release of the internally loaded VAP and antler growth factor.

[0084] (3) Biological significance of cascade triggering. The cascade response mechanism designed in this invention makes the drug release directly related to the severity of the lesion: the more severe the lesion → the higher the levels of MMP-2 and MMP-13 → the faster the degradation rate of the microsphere shell and core → the greater the VAP release. Experimental data confirm that in the dual-enzyme medium containing MMP-2 and MMP-13, the cumulative VAP release rate reached 79.3% in 24 hours, which is significantly higher than that in the single-enzyme medium (52.7% or 33.6%) and the enzyme-free medium (28.4%), with a release enhancement factor of 2.17 times. This intelligent design of on-demand drug release is one of the core innovations that distinguishes it from all existing topical patches.

[0085] IV. The principle of cartilage repair and nerve regeneration of active components in deer antler; As the principal ingredient in this invention, deer antler exhibits multi-target and multi-level biological effects of its active components: (1) Deer antler polypeptide (VAP) promotes the proliferation of nucleus pulposus cells and the synthesis of extracellular matrix. VAP (3-10 kDa) is rich in a variety of active peptides, which can promote the proliferation of nucleus pulposus cells and the synthesis of type II collagen (Col II) and aggrecan by activating the PI3K / Akt signaling pathway and the Wnt / β-catenin pathway.

[0086] (2) Deer antler polypeptide inhibits apoptosis and pyroptosis of nucleus pulposus cells. During intervertebral disc degeneration, excessive reactive oxygen species (ROS) and inflammatory factors activate the NLRP3 inflammasome, inducing pyroptosis of nucleus pulposus cells. VAP can inhibit the activation of the pyroptosis pathway by upregulating the expression of antioxidant enzymes and downregulating the levels of caspase-1 and GSDMD-N-terminus, thus protecting nucleus pulposus cells from programmed cell death.

[0087] (3) Natural growth factors in deer antler promote nerve root repair. The >10 kDa macromolecular active components of deer antler contain IGF-1 (insulin-like growth factor-1), TGF-β (transforming growth factor-β), and NGF (nerve growth factor). Among them, NGF can promote the recovery of sensory and motor function of compressed nerve roots, while IGF-1 synergistically promotes chondrocyte differentiation and nucleus pulposus repair. These macromolecular proteins are slowly released through the liposome transition layer and absorbed through the skin microcirculation before acting on the lesion area, realizing the TCM treatment concept of treating both the root cause and the symptoms by addressing both the tendons and bones.

[0088] V. ROS scavenging and microenvironment regulation mechanism of CeO2@PDA nanozymes;

[0089] Patients with lumbar disc herniation exhibit significant oxidative stress in the intervertebral disc. Compressed nucleus pulposus cells and infiltrating inflammatory cells produce large amounts of reactive oxygen species (ROS), including superoxide anions (O2·-), hydrogen peroxide (H2O2), and hydroxyl radicals (·OH). Excessive ROS attack nucleus pulposus cell membrane lipids, proteins, and DNA, exacerbating apoptosis and matrix degradation.

[0090] This invention introduces polydopamine-encapsulated cerium oxide nanozyme (CeO2@PDA) into an enzyme-responsive hydrogel controlled-release layer. Its ROS scavenging mechanism is based on the following mechanism: (1) Valence state cycling and SOD simulation activity of cerium oxide nanocrystals. CeO2 nanoparticles have Ce on their surface. 3+ / Ce 4+ A redox pair, and reversible conversion can occur between them. Ce 3+ It can react with superoxide anions to generate Ce. 4+ and H2O2, Ce 4+ It can also react with H2O2 to be reduced to Ce. 3+ It also releases O2. This cycle endows CeO2 superoxide dismutase (SOD) with similar activity.

[0091] (2) Synergistic antioxidant effect of polydopamine coating. Polydopamine (PDA) coating is rich in catechol groups, which can directly react with ROS such as hydroxyl radicals to undergo redox reactions. At the same time, it stabilizes CeO2 nanoparticles through hydrogen bonding and π-π stacking, preventing them from agglomerating and becoming inactive.

[0092] (3) ROS scavenging has multiple regulatory effects on the intervertebral disc microenvironment. By scavenging excess ROS, CeO2@PDA nanozymes can: reduce oxidative damage to nucleus pulposus cells and maintain mitochondrial membrane potential stability; inhibit the activation of the NF-κB signaling pathway and reduce the release of pro-inflammatory factors such as TNF-α and IL-1β; downregulate MMP expression and slow down the degradation rate of extracellular matrix, forming a synergistic effect with the pro-synthetic effect of VAP.

[0093] VI. Structural support and mechanical adaptation principle of triple interpenetrating network hydrogels; The enzyme-responsive hydrogel controlled-release layer of the plaster adopts a PVA / sodium alginate / GelMA triple interpenetrating network structure, and its design principle takes into account both mechanical properties and drug release function: (1) Freeze-thaw cycle to form a physical cross-linked network of PVA. The PVA solution is subjected to repeated freeze-thaw treatment (3 cycles of freezing at -20℃ and thawing at room temperature). The PVA molecular chains form microcrystalline regions through hydrogen bonds, constructing a physical cross-linked network framework, which gives the hydrogel good elasticity and toughness, and meets the conformal fitting requirements of waist movement.

[0094] (2) UV crosslinking forms a GelMA chemical network. The methacryloyl groups on the side chains of GelMA molecules undergo free radical polymerization under the action of photoinitiator LAP and 365 nm UV light to form a covalent crosslinked network. The presence of the GelMA network significantly improves the mechanical strength and dimensional stability of the hydrogel.

[0095] (3) Drug release regulation through calcium alginate ion cross-linking. Sodium alginate reacts with Ca in skin exudate. 2+ Ionic crosslinking occurs, forming calcium alginate gel. The crosslinking density of this network is affected by Ca. 2+ Concentration control allows for sustained drug release.

[0096] In summary, this invention, through the integration of a three-layer system, constructs a complete treatment chain encompassing transdermal penetration, a transitional reservoir, and intelligent responsive controlled release: the microneedle layer breaks through the physical barrier of the stratum corneum, directly delivering the active ingredients of deer antler and analgesic and anti-inflammatory components to the dermis for rapid onset of action. The liposome transitional reservoir layer provides secondary sustained release and protection, maintaining a drug concentration plateau for continuous supply. The enzyme-responsive hydrogel controlled release layer intelligently adjusts the drug release rate according to the pathological state of the lesion, achieving on-demand drug release. The active ingredients of deer antler promote the repair of nucleus pulposus cells and nerve regeneration, while CeO2@PDA nanoenzymes scavenge ROS and improve the microenvironment, achieving both symptomatic and root-cause treatment.

[0097] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. The present invention has been described in detail with reference to preferred embodiments. Those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications and substitutions should be covered within the scope of the present invention.

Claims

1. A method for preparing a traditional Chinese medicine plaster for lumbar disc herniation, characterized in that, Includes the following steps: S1, Pre-treatment of Chinese herbal raw materials: The Chinese herbal raw materials used include deer antler, drynaria, eucommia, angelica pubescens, chuanxiong, borneol, and menthol; S2, Graded extraction and processing of active components from deer antler: S2.1, Prepare VAP lyophilized powder; S2.2, Prepare freeze-dried powder of deer antler macromolecular active components; S2.3, prepare VAP nanoliposome suspension; S2.4, Preparation of borneol / menthol / β-cyclodextrin inclusion complex; S3, extracting and purifying other Chinese medicinal materials besides deer antler to prepare freeze-dried powder of Chinese medicinal compound extract; Preparation of S4, MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres: S4.1, Prepare MMP-2 responsive peptide crosslinking agent and MMP-13 responsive peptide crosslinking agent; S4.2 Preparation of core-shell structured dual-enzyme responsive microspheres: The microspheres consist of an outer shell layer and an inner core layer. The outer shell layer is prepared based on the MMP-2 responsive polypeptide cross-linking agent, and the inner core layer is prepared based on the MMP-13 responsive polypeptide cross-linking agent, VAP lyophilized powder, and lyophilized powder of deer antler macromolecular active components. The MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres are prepared by combining these components. S5, Preparation of CeO2@PDA nanozyme; S6, Fabrication of soluble hierarchical loaded microneedle array: S6.1, Prepare the microneedle mold; S6.2, Prepare the PVP / HA matrix solution; S6.3, Preparation of graded loading solutions: Drug loading solution in the needle tip region: Take the VAP nanoliposome suspension, add the borneol / menthol / β-cyclodextrin inclusion complex and PVP / HA matrix solution to obtain the drug loading matrix solution in the needle tip region; Drug solution in the middle of the needle body and drug solution at the base of the needle body: Take the freeze-dried powder of the Chinese herbal compound extract and add it to the PVP / HA matrix solution to obtain the drug-loaded matrix solution in the middle of the needle body and the drug-loaded matrix solution at the base of the needle body; S6.4, Preparation of microneedle array film: The drug-loaded matrix liquid in the needle tip area is filled into the needle tip part of the mold, the drug-loaded matrix liquid in the middle part of the needle body is filled into the middle part of the mold needle body, and the drug-loaded matrix liquid in the base part of the needle body is filled into the base part of the needle body. After drying, a microneedle array film is obtained. S7, Preparation of liposome transition reservoir layer: Prepare PVP / PVA solution, add the VAP nanoliposome suspension and the freeze-dried powder of the deer antler macromolecular active component to the PVP / PVA solution to obtain a mixture, and coat the mixture on the backing surface of the microneedle array membrane to form a liposome transition reservoir layer. S8, Preparation of enzyme-responsive hydrogel controlled-release layer: S8.1, Preparation of hydrogel prepolymer solution: Take PVA solution, sodium alginate solution, and GelMA solution, mix them, add photoinitiator, the freeze-dried powder of the Chinese herbal compound extract, the CeO2@PDA nanozyme and the MMP-2 / MMP-13 dual enzyme cascade response microspheres to prepare hydrogel prepolymer solution. S8.2, the hydrogel prepolymer solution is poured onto the microneedle array membrane, and after treatment, an enzyme-responsive hydrogel controlled-release layer is formed.

2. The method for preparing the traditional Chinese medicine plaster for lumbar disc herniation according to claim 1, characterized in that, The following Chinese medicinal materials are used in S1 by weight: 30 parts deer antler, 15 parts drynaria rhizome, 12 parts eucommia bark, 10 parts angelica pubescens root, 8 parts chuanxiong rhizome, 3 parts borneol, and 2 parts menthol.

3. The method for preparing the traditional Chinese medicine plaster for lumbar disc herniation according to claim 1, characterized in that, The specific steps for preparing VAP lyophilized powder in S2.1 are as follows: Fresh deer antler was freeze-dried and then pulverized to obtain freeze-dried deer antler powder. The freeze-dried deer antler powder was added to pre-cooled PBS buffer, and then papain was added for enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the temperature was raised to inactivate the papain. After centrifugation, the supernatant was collected. The supernatant was sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 10 kDa and 3 kDa for fractionation to obtain VAP fractions with molecular weight ranges of 3–10 kDa. The retentate from the 3 kDa ultrafiltration membrane was collected and freeze-dried to obtain VAP freeze-dried powder. The specific steps for preparing the freeze-dried powder of deer antler macromolecular active components in step S2.2 are as follows: The retentate from the 10 kDa ultrafiltration membrane in S2.1 was collected and freeze-dried to obtain freeze-dried powder of deer antler macromolecular active components.

4. The method for preparing the traditional Chinese medicine plaster for lumbar disc herniation according to claim 1, characterized in that, The specific steps for preparing the VAP nanoliposome suspension in S2.3 are as follows: Take egg yolk lecithin and cholesterol and place them in a container. Add anhydrous ethanol and dissolve them completely. Then, evaporate them under vacuum until a uniform and transparent lipid film is formed on the container wall. Take the VAP lyophilized powder, dissolve it in PBS buffer, and prepare a VAP solution; VAP solution is added to the container, glass beads are added, and the mixture is rotated to hydrate and detach the lipid membrane, forming a liposome suspension. The liposome suspension was subjected to probe sonication under ice bath conditions, and then granulated through a filter membrane to obtain VAP nanoliposome suspension.

5. The method for preparing the traditional Chinese medicine plaster for lumbar disc herniation according to claim 1, characterized in that, The specific steps for preparing the MMP-2 responsive peptide crosslinking agent and the MMP-13 responsive peptide crosslinking agent in step S4.1 are as follows: First, MMP-2 responsive peptide precursors and MMP-13 responsive peptide precursors with amino linkages at both ends were prepared by solid-phase peptide synthesis. The recognition sequence of the MMP-2 responsive peptide is GPLGIAGQ, and the recognition sequence of the MMP-13 responsive peptide is PQGLA. Then, N-acryloyloxysuccinimide ester was used to acryloylate the amino groups at both ends of the MMP-2 responsive peptide precursor and the MMP-13 responsive peptide precursor, respectively, to obtain the MMP-2 responsive peptide crosslinking agent Acryloyl-GPLGIAGQ-Acryloyl and the MMP-13 responsive peptide crosslinking agent Acryloyl-PQGLA-Acryloyl.

6. The method for preparing the traditional Chinese medicine plaster for lumbar disc herniation according to claim 5, characterized in that, The specific steps for preparing the core-shell dual-enzyme responsive microspheres in S4.2 are as follows: Take polyethylene glycol diacrylate, add Acryloyl-GPLGIAGQ-Acryloyl and a photoinitiator to form a shell prepolymer liquid; A glass capillary microfluidic device was used to form W / O / W double droplets with polyvinyl alcohol aqueous solution as the inner phase, shell prepolymer liquid as the outer phase, and mineral oil containing Tween 20 as the collecting phase. The shell was photocrosslinked and cured by ultraviolet light irradiation to obtain hollow microspheres. Sodium alginate was added to Acryloyl-PQGLA-Acryloyl, and after dissolution, the VAP lyophilized powder and the deer antler macromolecular active component lyophilized powder were added to serve as the core prepolymer solution. Hollow microspheres were immersed in a core prepolymer solution to allow the solution to fully penetrate the hollow cavity of the microspheres. The microspheres were then removed and immersed in a calcium chloride solution for cross-linking and curing at room temperature. Finally, they were photocross-linked with ultraviolet light to obtain MMP-2 / MMP-13 dual-enzyme cascade responsive microspheres.

7. The method for preparing the traditional Chinese medicine plaster for lumbar disc herniation according to claim 1, characterized in that, The specific steps for preparing CeO2@PDA nanozymes in S5 are as follows: Cerium nitrate hexahydrate is dissolved in water, and ammonia is added to adjust the pH to alkaline, causing cerium ions to precipitate and yielding the cerium hydroxide precursor. Cerium hydroxide precursor was calcined and then cooled to room temperature to obtain cerium oxide nanoparticles; Cerium oxide nanoparticles were dispersed in Tris-HCl buffer, dopamine hydrochloride was added, the precipitate was collected by centrifugation, and the CeO2@PDA nanozyme was obtained by freeze-drying.

8. The method for preparing the traditional Chinese medicine plaster for lumbar disc herniation according to claim 1, characterized in that, The specific operation of S8.2 is as follows: The microneedle array membrane is laid flat at the bottom of the mold with the microneedle tips facing down. The hydrogel prepolymer obtained in S8.1 is poured onto the microneedle array membrane. The mold is then frozen, removed, and thawed at room temperature. The freeze-thaw cycle is repeated multiple times to form a PVA physical crosslinking network. After the freeze-thaw cycle is completed, the mold is placed under a UV lamp to irradiate it, causing the GelMA to undergo photocrosslinking and form a chemical crosslinking network, and finally the mold is demolded.

9. A traditional Chinese medicine plaster for lumbar disc herniation, characterized in that, It is prepared using the method described in any one of claims 1 to 8 for the preparation of traditional Chinese medicine plaster for lumbar disc herniation.

10. The traditional Chinese medicine plaster for lumbar disc herniation according to claim 9, characterized in that, The front of the plaster has a medical breathable non-woven fabric backing layer, and the back of the plaster has a release protective layer.

Images