Azo calixarene-based broad-spectrum quantitative drug-loading acupuncture needle as well as preparation method and application thereof

Abstract

The invention belongs to but is not limited to the technical field of Chinese and western combination, and discloses an azo calixarene-based broad-spectrum quantitative drug-loading acupuncture needle and a preparation method and application thereof, and the strong interaction between silver and sulfydryl is utilized to realize the modification of sulfydryl azo calixarene on the surface of a silver needle; based on interaction of subjects and objects, the modified silver needle is soaked in various medicine solutions, and the medicine-carrying acupuncture needle is obtained. Needles and medicines are combined, 'same-point administration 'can be realized, on one hand, specific acupuncture points are stimulated through acupuncture machinery to play a treatment effect, on the other hand, the medicine effect of the medicines is played, and the synergistic interaction effect of acupuncture point mechanical stimulation and the medicine curative effect is realized. Meanwhile, the prepared medicine-carrying acupuncture needle can effectively screen the combination of acupuncture points and medicines, so that the optimal treatment scheme is screened out, and the treatment effect is improved. According to the medicine-carrying acupuncture needle with the broad-spectrum quantification function, an efficient and convenient tool is provided for accurately screening the optimal combination of acupuncture points and medicines.

Description

Technical Field

[0001] The present invention belongs to but is not limited to the field of combining traditional Chinese and Western medicine, and in particular relates to a broad-spectrum quantitative drug-loaded acupuncture needle based on azocalixarene and a preparation method and application thereof. Background Art

[0002] Acupuncture, as an important treatment method in traditional Chinese medicine, is deeply favored by people for its advantages such as small side effects, small trauma, low cost and wide application of diseases. In clinical treatment, acupuncture is often combined with other therapies such as drug therapy and surgery to improve the comprehensive treatment effect. Among them, the combination of acupuncture and medicine can not only give play to their respective advantages and achieve synergistic effects, but also effectively reduce the toxic side effects of drugs. Faced with the choice of many acupoints and drugs, in order to maximize clinical benefits, accurately screening the best combination of acupoints and drugs is the key to improving the treatment effect. The prerequisite for solving this problem is to quantitatively load various drugs onto acupuncture needles. However, this process still faces huge challenges.

[0003] The prior art provides a drug-coated acupuncture needle, which is prepared by directly coating the drug on the needle surface. The drug is mainly fixed on the needle surface by physical adsorption and is often used for local drug release therapy. This type of acupuncture needle mainly relies on the weak interaction between the needle and the drug to fix the drug, resulting in uneven drug release and limited drug loading.

[0004] Existing technical problems: 1) Unstable drug binding: In the existing technology, the drug and the needle surface mainly rely on physical adsorption, which is easy to fall off during storage and use, affecting the stability and efficacy of drug delivery.

[0005] 2) Limited types of loaded drugs: Due to the lack of specific chemical action sites, existing technologies have poor adaptability to the types and properties of drugs, which limits the loading capacity of multiple drugs.

[0006] 3) The release rate is difficult to control: physical adsorption leads to uneven drug release rate, which makes it impossible to achieve sustained release effect or broad-spectrum application, affecting the therapeutic effect.

[0007] 4) Low efficiency of the preparation process: The traditional direct coating method is difficult to evenly cover the needle surface, resulting in low preparation efficiency and poor product consistency. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides a broad-spectrum quantitative drug-loaded acupuncture needle based on azocalixarene and a preparation method and application thereof, which provides an efficient and convenient tool for accurately screening the optimal combination of acupoints and drugs.

[0009] The present invention is achieved by a method for preparing a broad-spectrum quantitative drug-loaded acupuncture needle based on azocalixarene, which utilizes the strong interaction between silver and thiol to modify the surface of the silver needle with thiol azocalixarene; based on the host-guest interaction, the modified silver needle is immersed in various drug solutions to obtain the drug-loaded acupuncture needle; the method specifically comprises the following steps: Step 1, synthesizing mercaptoazocalixarene SAC4A-SH and preparing a SAC4A-SH solution; Step 2, activating the surface of the silver needle to remove oxides on the surface of the silver needle; Step 3, immersing the activated silver needle in a SAC4A-SH solution, using the strong interaction between silver and thiol, SAC4A-SH is firmly attached to the surface of the silver needle, thereby obtaining a SAC4A-SH-modified silver needle with drug loading capacity (SAC4A-SH-silver needle); Step 4: soak the modified silver needle in a drug solution to obtain a drug-loaded acupuncture needle.

[0010] Furthermore, the concentration of the SAC4A-SH solution in step 1 is 5 mM.

[0011] Further, the synthesis method of the SAC4A-SH is as follows: (1) Add 690 mg NH2-PEG-CSAC4A (0.57 mmol) to a 100 mL round-bottom flask and dissolve it in 40 mL phosphate buffer; (2) Add 94 mg of 2-iminothiolane hydrochloride (0.68 mmol) into a round-bottom flask and stir the mixture in the dark at room temperature under nitrogen protection for 12 hours; (3) After the reaction was completed, the product was placed in a dialysis bag with a molecular weight of 500 and dialyzed in water in the dark for 48 hours. After the dialysis was completed, the solvent was dried in a vacuum to obtain 530 mg of the product with a yield of 66%.

[0012] Further, the preparation method of the NH2-PEG-CSAC4A is as follows: (1) Add 200 mg of monocarboxytrisulfonate azocalix[4]arene (0.18 mmol) to a 100 mL round-bottom flask and dissolve it in 25 mL of N,N-dimethylformamide; (2) Add 44 mg of N,N-diisopropylethylamine (0.34 mmol) and 78 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.21 mmol) into a round-bottom flask and stir at room temperature for 30 minutes; add 45 mg of [2-(2-aminoethoxy)ethyl]carbamic acid tert-butyl ester (0.22 mmol) into the above reaction system and stir at room temperature for 12 hours; vacuum spin dry the solvent to obtain a red powder; (3) The solid was added to a 100 mL round-bottom flask, dissolved with 40 mL of dimethyl sulfoxide, and then 3 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 6 hours. The solvent was dried under vacuum, and the product was recrystallized from water and methanol to obtain 138 mg of the product with a yield of 60%.

[0013] Furthermore, the soaking time in step 2 is 24 to 72 hours.

[0014] Furthermore, the method for activating the surface of the silver needle in step 2 includes: The silver needle was polished with Al2O3 suspension, ultrasonically cleaned with anhydrous ethanol and ultrapure water for 2 min each, then immersed in 6M nitric acid solution for a few seconds, mechanically scrubbed on suede, and ultrasonically cleaned with anhydrous ethanol and ultrapure water for 2 min each, with N2 protection throughout the process.

[0015] Furthermore, the concentration of the drug solution in step three is 0.5-5 mM, and the loading time each time is 10-50 min.

[0016] The invention provides a drug-loaded acupuncture needle, comprising a silver needle substrate and a SAC4A-SH coating loaded on the surface thereof, wherein the coating is firmly combined with the silver needle surface through a strong interaction of thiol groups and can uniformly adsorb and release drug molecules, and is suitable for broad-spectrum drug delivery and quantitative release.

[0017] Furthermore, the acupuncture needle is prepared by the following method: (1) Activate the surface of the silver needle substrate to remove oxides; (2) Soaking the activated silver needle in a SAC4A-SH solution to allow SAC4A-SH to be firmly attached to the surface of the silver needle via the thiol group; (3) Soak the modified silver needles in a drug solution to load the target drug molecules.

[0018] Furthermore, the SAC4A-SH coating has a highly adjustable drug loading capacity, with an attached drug concentration of 0.5-5 mM, and has a sustained-release function, which can stably release drugs during use.

[0019] Furthermore, the surface activation method of the silver needle substrate includes mechanical polishing using Al2O3 suspension, followed by ultrasonic cleaning using anhydrous ethanol and ultrapure water, and short immersion in 6M nitric acid solution, followed by scrubbing with suede, and finally drying under N2 protection to ensure surface cleaning and enhance the adhesion effect of SAC4A-SH.

[0020] The present invention utilizes the easy modification, broad-spectrum and efficient drug loading characteristics of azocalixarene to prepare an acupuncture needle with broad-spectrum quantitative drug loading capacity; thiol-modified azocalixarene is designed based on azocalixarene. It is not only easy to modify on the surface of silver needles in a simple way, but also can carry out broad-spectrum quantitative loading of drugs. The needle and the drug are combined to realize "drug administration at the same acupoint" to achieve a synergistic effect. Through the strong interaction between silver and thiol, the thiol-modified azocalixarene can be firmly attached to the surface of the silver needle, thereby realizing the ability to quantitatively load drugs on the acupuncture needle, meeting the needs of accurately screening the best acupoints and drug combinations; the prepared drug-loaded acupuncture needle can effectively screen the combination of acupoints and drugs, thereby screening out the optimal treatment plan to improve the treatment effect. The drug-loaded acupuncture needle with broad-spectrum quantitative function provided by the present invention provides an efficient and convenient tool for accurately screening the best combination of acupoints and drugs.

[0021] The present invention selected three acupoints (Zusanli, Yanglingquan and Dubi) and three drugs (triamcinolone acetonide, celecoxib and lidocaine) to create a 3×3 array, and used rat osteoarthritis as an experimental model to verify the array. The experimental results showed that the treatment scheme of loading drugs on acupuncture needles showed significant differences in relieving osteoarthritis in rats compared with acupuncture alone and drug treatment alone, indicating a synergistic effect. In addition, broad-spectrum quantitative drug-loaded acupuncture needles can be used to screen the best combination of acupoints and drugs to maximize the therapeutic effect. And the drug-loaded acupuncture needles of the present invention have good biosafety and are expected to provide a new treatment strategy for the treatment of osteoarthritis.

[0022] Second, the existing technology involves the drug loading method and preparation process of acupuncture needles, and the main technical problems are as follows: 1) Poor drug adhesion stability: In the existing technology, drugs are mainly fixed on the surface of acupuncture needles by physical adsorption. The drugs are easy to fall off or released too quickly, making it difficult to ensure a stable supply of drugs during treatment.

[0023] 2) Low loading efficiency: Physical adsorption or direct coating methods are difficult to achieve uniform loading, and drug utilization is low, which limits the application of acupuncture needles in efficient treatment.

[0024] 3) Limited drug types: Existing technologies are difficult to adapt to the broad-spectrum loading of different types of drugs, especially drugs with certain special molecular structures.

[0025] 4) The release rate is difficult to control: Due to the lack of a controllable binding mechanism, existing acupuncture needles are difficult to achieve quantitative and sustained drug release.

[0026] Technical problems solved by the present invention: The present invention provides a broad-spectrum quantitative drug-loaded acupuncture needle based on azocalixarene and a preparation method thereof, which significantly solves the above technical problems: 1) Improve the stability of drug attachment: Utilize the strong interaction between the thiol group in the SAC4A-SH molecule and the surface of the silver needle to form a strong chemical bond, making the drug binding more stable and avoiding drug shedding during storage or use.

[0027] 2) Improve drug loading efficiency: Enhance the binding ability between drugs and coatings through the host-guest interaction of azocalix arene, increase drug loading, and ensure the uniformity of the coating.

[0028] 3) Adaptability to broad-spectrum drug loading: SAC4A-SH has diverse molecular recognition capabilities and can adapt to the loading of a variety of drug molecules, especially for drugs with complex structures or large molecular weights.

[0029] 4) Achieve sustained-release drug release: By regulating the coating thickness and drug loading time, the present invention achieves quantitative loading and sustained-release of drugs, significantly improving the therapeutic effect.

[0030] Significant technological advances achieved: 1) Simplified preparation process: The present invention adopts molecular self-assembly technology, which significantly simplifies the preparation process of acupuncture needle coating, improves the preparation efficiency, and is suitable for large-scale production.

[0031] 2) Performance optimization: The drug-loaded acupuncture needles of the present invention have high drug loading capacity, stability and controllable release rate, meeting the needs of complex medical scenarios.

[0032] 3) Broad-spectrum adaptability: By designing the molecular structure of azocalix arene, the present invention can load a variety of drug molecules to meet a wide range of treatment needs, broadening the application prospects of acupuncture needles in precision medicine.

[0033] 4) Environmental friendliness: The raw materials and operating conditions in the preparation process are environmentally friendly and pollution-free, meeting the requirements of modern green manufacturing.

[0034] In summary, the present invention is significantly superior to the prior art in terms of improving drug loading efficiency, binding stability and release controllability, and solves the key bottleneck problem of acupuncture needles in drug delivery applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 The present invention provides a flowchart of a method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene.

[0036] Figure 2 It is the hydrogen nuclear magnetic resonance spectrum of NH2-PEG-CSAC4A provided in the embodiment of the present invention.

[0037] Figure 3 It is the ESI mass spectrum of NH2-PEG-CSAC4A provided in the embodiment of the present invention.

[0038] Figure 4 It is the hydrogen nuclear magnetic resonance spectrum of SAC4A-SH provided in the embodiment of the present invention.

[0039] Figure 5 It is the carbon nuclear magnetic resonance spectrum of SAC4A-SH provided in the embodiment of the present invention.

[0040] Figure 6 It is the ESI mass spectrum of SAC4A-SH provided in the embodiment of the present invention.

[0041] Figure 7 It is the amount of SAC4A-SH modified on the acupuncture needle provided in the examples of the present invention.

[0042] Figure 8 The invention discloses an indicator replacement fluorescence titration method for determining the complexation of sulfonated azocalix[4]arene (SAC4A) with a drug (triamcinolone acetonide).

[0043] Fig. 9 The guest replacement fluorescence titration method provided in the embodiment of the present invention is used to determine the complexation of SAC4A to the drug (celecoxib).

[0044] Fig.10 The guest replacement fluorescence titration method provided in the embodiment of the present invention is used to determine the complexation of SAC4A to the drug (lidocaine).

[0045] Fig.11 It is the amount of drug loaded in SAC4A-SH-silver needle provided in the embodiment of the present invention.

[0046] Fig.12 This is the behavioral evaluation of each group of rats after treatment provided in the embodiments of the present invention.

[0047] Fig.13 It is the expression of each inflammatory factor in each group of rats after treatment provided in the embodiments of the present invention.

[0048] Fig.14 This is the behavioral evaluation of rats after treatment with the 3'3 array provided in the embodiment of the present invention.

[0049] Fig.15 The expression of various inflammatory factors in rats after treatment with the 3'3 array provided in the embodiment of the present invention.

[0050] Fig.16 This is a hematoxylin-eosin and safranin fast green staining image of a 3′3 array rat after treatment provided by an embodiment of the present invention.

[0051] Fig.17 These are the blood routine and blood biochemical results of each group of rats for safety evaluation provided in the embodiments of the present invention.

[0052] Fig.18 This is a hematoxylin-eosin staining image of the heart, liver, spleen, lung, and kidney of each group of rats for safety evaluation provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0053] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0054] The technical problems that the solution of the present invention can solve are: Improved drug binding stability: Through the strong interaction between SAC4A-SH and the thiol-silver on the surface of the silver needle, a stable chemical binding site is provided, avoiding the problem of easy drug shedding.

[0055] Broad-spectrum drug loading capacity: SAC4A-SH coating has a unique calixarene structure, which can bind a variety of drug molecules through host-guest interactions and is suitable for loading a broad spectrum of drugs.

[0056] Controlled drug release: By regulating the thickness of the coating and the concentration of the drug solution, quantitative drug loading and sustained release can be achieved to improve the therapeutic effect.

[0057] The preparation process is efficient and uniform: solution processing and molecular self-assembly methods are used to form a uniform coating on the needle surface, significantly improving preparation efficiency and product consistency.

[0058] In summary, this new solution is significantly superior to the existing technology in terms of drug binding stability, loading range, release control and preparation efficiency, and solves several key problems in the existing technology.

[0059] like Figure 1 As shown, the embodiment of the present invention provides a method for preparing acupuncture needles with broad spectrum quantitative drug loading based on azocalixarene, which utilizes the strong interaction between silver and thiol to modify the surface of the silver needle with thiol azocalixarene; based on the host-guest interaction, the modified silver needle is immersed in various drug solutions to obtain the drug-loaded acupuncture needle; the method specifically comprises the following steps: Step 1, synthesizing mercaptoazocalixarene SAC4A-SH and preparing a SAC4A-SH solution; Step 2, activating the surface of the silver needle to remove oxides on the surface of the silver needle; Step 3, immersing the activated silver needle in a SAC4A-SH solution, using the strong interaction between silver and thiol, SAC4A-SH is firmly attached to the surface of the silver needle, thereby obtaining a SAC4A-SH-modified silver needle with drug loading capacity (SAC4A-SH-silver needle); Step 4: soak the modified silver needle in a drug solution to obtain a drug-loaded acupuncture needle.

[0060] Example 1: Synthesis of SAC4A-SH ; (1) Synthesis of monocarboxytrisulfonate azocalix[4]arene (1c) Monocarboxytrisulfonic acid calixtetraarene was synthesized according to the scheme of the literature [SY Yao, K. Cai, DS Guo, Acta Polym. Sin., 2022, 53,1271-1278.].

[0061] (2) Synthesis of NH2-PEG-CSAC4A (3a) 200 mg of monocarboxy trisulfonate azocalix[4]arene (0.18 mmol) was added to a 100 mL round-bottom flask and dissolved in 25 mL of N,N-dimethylformamide. Then 44 mg of N,N-diisopropylethylamine (0.34 mmol) and 78 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.21 mmol) were added to the round-bottom flask and stirred at room temperature for 30 minutes. 45 mg of tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (0.22 mmol) was added to the above reaction system and stirred at room temperature for 12 hours. The solvent was dried under vacuum to obtain a red powder. The solid was added to a 100 mL round-bottom flask, dissolved with 40 mL of dimethyl sulfoxide, and then 3 mL of trifluoroacetic acid was added, and stirred at room temperature for 6 hours; the solvent was dried under vacuum, and the product was recrystallized from water and methanol to obtain 138 mg of the product with a yield of 60%.

[0062] like Figure 2 As shown, the compound NH2-PEG-CSAC4A 1 H NMR (400 MHz, DMSO- d 6) δ: 7.94 (d, J = 8.5 Hz, 2H), 7.85 (t, J = 6.9 Hz, 10H), 7.74 (d, J= 5.7 Hz, 12H), 4.11(m, 8H), 3.59 3.37 (m, 8H), 2.86 (s, 2H) ppm. like Figure 3 As shown, the Mass spectrum (ESI) of compound NH2-PEG-CSAC4A: [M+H] + : m / z calcd. for (C 57 H 51 N 10 O 15 S3 + ): 1211.26, found: 1211.26817. (3) Synthesis of SAC4A-SH (3e) 690 mg NH2-PEG-CSAC4A (0.57 mmol) was added to a 100 mL round-bottom flask and dissolved in 40 mL phosphate buffer. 94 mg 2-iminothiolane hydrochloride (0.68 mmol) was added to the round-bottom flask and stirred for 12 hours at room temperature in the dark under nitrogen protection. After the reaction was completed, the mixture was placed in a dialysis bag with a molecular weight of 500 and dialyzed in water in the dark for 48 hours. After the dialysis was completed, the solvent was dried in a vacuum to obtain 530 mg of SAC4A-SH with a yield of 66%.

[0063] like Figure 4 As shown, the compound SAC4A-SH 1 H NMR (400 MHz, DMSO- d 6) δ: 7.94 (d, J =7.5 Hz, 2H), 7.86 7.79 (m, 10H,), 7.73 7.61 (m, 12H), 4.09 3.91 (m, 8H), 3.573.37 (m, 8H), 1.35 (d, J = 4.6 Hz 6H), 1.23 (s, 1H) ppm. like Figure 5 As shown, the compound SAC4A-SH 13 C NMR (100 MHz, DMSO- d6) δ: 170.84,167.28, 158.88, 157.17, 155.08, 152.40, 149.60, 145.28, 145.07, 137.83,137.10, 137.04, 136.94, 136.80, 131.75, 127.01, 124.86, 124.43, 124.35,122.34, 121.97, 71.55, 70.35, 44.86, 43.66, 34.79, 24.95, 22.75, 18.95 ppm. like Figure 6 As shown, the Mass spectrum (ESI) of compound SAC4A-SH: [M-2Na+3H] + : m / z calcd. for (C 61 H 55 C1N 11 NaO 15 S4 + ): 1368.36, found: 1368.36243. Example 2: Modification of SAC4A-SH on the surface of acupuncture needles First, the silver needle was polished with Al2O3 suspension paste, ultrasonically cleaned with anhydrous ethanol and ultrapure water for 2 minutes each, then immersed in 6M nitric acid solution for a few seconds, mechanically scrubbed on suede, and ultrasonically cleaned with anhydrous ethanol and ultrapure water for 2 minutes each, with N2 protection throughout the process. The pretreated silver needle was then immersed in a 5M SAC4A-SH solution deoxygenated with N2 for 48 hours. After that, it was taken out and rinsed with ultrapure water, and dried for later use.

[0064] Example 3: Amount of SAC4A-SH Modified on Acupuncture Needles Soak multiple silver needles in a 5mM, 500uL volume of SAC4A-SH solution. After soaking, take out the silver needles, wash them with water to remove the SAC4A-SH physically adsorbed on the needles, and add the washing solution to the soaked SAC4A-SH solution to make it fixed to a certain volume. By comparing the changes in the content of the SAC4A-SH solution before and after the silver needles are soaked, the total amount of SAC4A-SH modified on the silver needles is calculated using ultraviolet light, and finally the total amount is divided by the number of silver needles to obtain the amount of SAC4A-SH modified on each needle. The amount of SAC4A-SH modified on the silver needles is shown in Figure 7 .

[0065] Example 4: Binding constants of SAC4A to three drugs The binding constant was determined by competitive fluorescence titration (including indicator displacement titration and guest displacement titration) according to the method in [Yue, Y.; Lin, Y.; Chen, M.; Tian, ​​H.; Ma, R.; Wang, Z.; Chen, F.; Pan, Y.; Guo, D.; Azocalixarenes: a scaffold of universal excipients withhigh efficiency. Sci. China Chem. 2024, 67, 1697–1706.].

[0066] Determination of the bonding constant of SAC4A and triamcinolone acetonide: The mixed solution containing rhodamine B, SAC4A and triamcinolone acetonide was added successively to a quartz cuvette containing 2.5 mL of a mixed solution of rhodamine B and SAC4A. In order to keep the concentrations of rhodamine B and SAC4A constant during the titration process, the concentrations of SAC4A and rhodamine B in the mixed solution and the quartz cuvette were the same. The fluorescence intensity was tested before the first drop and after each drop. Figure 8 As shown, according to the 1:1 competition binding model, the binding constant value of triamcinolone acetonide and SAC4A was obtained by fitting the fluorescence intensity and drug concentration at its emission wavelength.

[0067] Determination of the binding constant between SAC4A and the guest (celecoxib and lidocaine): A mixed solution containing rhodamine B, SAC4A and the guest was added successively to a solution containing SAC4A and the guest with unknown binding constants. In order to keep the concentrations of the guest and SAC4A constant during the titration process, the concentrations of SAC4A and the guest in the mixed solution and the quartz cuvette were the same. The fluorescence intensity was tested before the first addition and after each drop. The fluorescence intensity of the drug at its emission wavelength and the concentration of rhodamine B were fitted according to the 1:1 competitive binding model to obtain the binding constant value of the guest and SAC4A (the binding constant of SAC4A and celecoxib is shown in Fig. 9 The binding constant of SAC4A and lidocaine is shown in Fig.10 ) Example 5: SAC4A-SH-silver needle loaded drug The prepared SAC4A-SH-silver needles were repeatedly immersed in water with a concentration of 0.5-5 mM, and the loading time was 10-50 min each time to obtain drug-loaded acupuncture needles. Fig.11 .

[0068] Example 6: Synergistic Effect of SAC4A-SH-Silver Needle Loaded with Triamcinolone Acetonide on Acupuncture Point Zusanli in the Treatment of Osteoarthritis After successful modeling, treatment was started once a day and ended after 28 days. The therapeutic effect was comprehensively evaluated by behavioral evaluation, including Lequesne MG score and hot plate pain threshold test, as well as the detection of inflammatory factor expression in the joint cavity.

[0069] (1) According to the evaluation criteria of the Lequesne MG scoring scale, the behavioral performance of rats was divided into four aspects: pain, joint range of motion, gait, and joint swelling. The specific scores were scored according to the Lequesne scale, and the scores of each item were recorded for comprehensive evaluation. The Lequesne MG scores of rats in each group are shown in Fig.12 a.

[0070] (2) One week before the start of the experiment, the rats were placed in plexiglass cages for half an hour every day to adapt to the experimental environment. The pain threshold of the rats was determined by observing the latency of the nociceptive response. The rats were placed on the surface of a metal hot plate with a set temperature of 55 ± 0.5 °C, and the plexiglass cages were made to meet the requirements of the hot metal surface. The timing was started as soon as the rat was placed on the hot plate, and the latency of the first licking of the hind paw was recorded. Each rat repeated the above steps three times and the average value was calculated. The measurement interval for the same rat should not be less than 40 minutes. Before each rat starts the experiment, the temperature of the metal plate should be matched with the temperature in the test room, and the surface of the test plate should be cleaned to reduce the influence of biological signals and urine on the experimental results. The pain thresholds of each group of rats can be found in Fig.12 b.

[0071] (3) Collect the synovial fluid from the joint cavity and centrifuge it at 3000 rpm for 10 minutes to obtain the test sample. Use a 0.45 mm filter membrane to remove impurities and use the ELISA method to detect the expression level of inflammatory factors (IL-1β and TNF-α) in the rat joint cavity. The expression results of inflammatory factors in the joint cavity of each group of rats are shown in Fig.13 .

[0072] Example 7: SAC4A-SH-silver needle as an effective tool for drug and acupoint screening After successful modeling, treatment was started, once a day, and ended after 28 days. Three acupoints (Zusanli, Yanglingquan, and Dubi) and three drugs (triamcinolone acetonide, celecoxib, and lidocaine) were selected to create a 3 × 3 array. The therapeutic effect was comprehensively evaluated through behavioral evaluation, including Lequesne MG score, hot plate pain threshold test, detection of inflammatory factor expression in the joint cavity, hematoxylin-eosin staining, and safranin fast green staining.

[0073] The Lequesne MG score, hot plate pain threshold test, and the test methods for the expression of inflammatory factors in the joint cavity are shown in (1), (2), and (3) of Example 5. The Lequesne MG score and pain threshold of each group of rats are shown in Fig.14 The expression results of inflammatory factors in the joint cavity are shown in Fig.15 .

[0074] The rat knee joint tissue was first fixed in 4% paraformaldehyde for 24 hours. After fixation, the samples were decalcified using 10% ethylenediaminetetraacetic acid decalcification solution. Next, the samples were dehydrated, waxed, and sagittally embedded, and finally sliced ​​to 3 microns thick. After slicing, hematoxylin-eosin and safranin fast green staining were used for staining. Finally, the staining of the knee cartilage sections was observed under an optical microscope at a magnification of 200 times. The hematoxylin-eosin and safranin fast green staining results of the rats in each group are shown in Fig.16 .

[0075] Example 8: Safety Assessment (1) Blood samples were collected from each group of rats for routine blood test and blood biochemistry test. The results of routine blood test and blood biochemistry test of each group of rats are shown in Fig.17 a and 17b.

[0076] (2) The main organs of rats in each group were collected for hematoxylin-eosin staining. The results of hematoxylin-eosin staining of the heart, liver, spleen, lung, and kidney of rats in each group are shown in Fig.18 .

[0077] Example 1: Azocalixarene-based anti-inflammatory drug-loaded acupuncture needles Acupuncture needles loaded with anti-inflammatory drugs (such as ibuprofen) are prepared to relieve local inflammation and pain.

[0078] Preparation steps: 1. Synthesis of SAC4A-SH: The mercaptoazocalixarene (SAC4A-SH) is prepared according to the steps of claim 3 and claim 4.

[0079] A 5 mM SAC4A-SH solution was prepared.

[0080] 2. Silver needle surface activation: Use Al2O3 suspension to polish the surface of the silver needle.

[0081] Ultrasonic cleaning was performed with anhydrous ethanol and ultrapure water for 2 minutes each.

[0082] The silver needles were immersed in 6M nitric acid solution for a few seconds, then mechanically scrubbed and cleaned again, all under nitrogen protection.

[0083] 3. Coating modification: The activated silver needles were immersed in 5 mM SAC4A-SH solution for 48 hours.

[0084] The surface of the silver needle forms a stable bond with silver through the thiol group to form a SAC4A-SH coating.

[0085] 4. Drug loading: The modified silver needles were immersed in 1 mM ibuprofen solution and the loading time was 30 min.

[0086] The drug-loaded acupuncture needles with sustained-release anti-inflammatory effects are obtained.

[0087] Test results: The drug release stability experiment showed that the acupuncture needle could continuously release ibuprofen within 12 hours and was suitable for local anti-inflammatory treatment.

[0088] In animal experiments, it significantly reduced local inflammation and pain responses.

[0089] Example 2: Acupuncture needles loaded with anti-tumor drugs based on azocalixarene Acupuncture needles loaded with anti-tumor drugs (such as paclitaxel) are prepared for adjuvant treatment of cancer patients.

[0090] Preparation steps: 1. Synthesis of SAC4A-SH: The method described in claim 3 and claim 4 was used to synthesize mercaptoazocalixarene (SAC4A-SH).

[0091] A 5 mM SAC4A-SH solution was prepared.

[0092] 2. Silver needle surface activation: The surface of the silver needle is activated according to the method described in claim 6 to ensure that the surface is clean and free of oxides.

[0093] 3. Coating modification: The activated silver needles were immersed in 5 mM SAC4A-SH solution for 72 hours.

[0094] SAC4A-SH forms a strong bond with the silver surface via the thiol groups.

[0095] 4. Drug loading: The modified silver needles were immersed in 2 mM paclitaxel solution and the loading time was 40 min.

[0096] The drug-loaded acupuncture needles with anti-tumor sustained-release function are obtained.

[0097] Test results: Drug release experiments showed that acupuncture needles slowly released paclitaxel within 24 hours, and the amount of drug released was stable and controllable.

[0098] In a tumor mouse model, drug-loaded acupuncture needles showed strong tumor inhibitory effects while reducing chemotherapy side effects.

[0099] The above examples respectively focus on anti-inflammatory and anti-tumor application scenarios, demonstrating the adaptability and technical advantages of the acupuncture needles of the present invention under different drug loading conditions. These products have the characteristics of stable drug release and significant therapeutic effects, and are suitable for a wide range of clinical needs.

[0100] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene, characterized in that: The following steps are involved: Step 1, synthesizing mercaptoazocalixarene SAC4A-SH and preparing a SAC4A-SH solution; Step 2, activating the surface of the silver needle to remove oxides on the surface of the silver needle; Step 3, immersing the activated silver needle in a SAC4A-SH solution, using the strong interaction between silver and thiol, SAC4A-SH is firmly attached to the surface of the silver needle, thereby obtaining a SAC4A-SH-modified silver needle with drug loading capacity; Step 4: soak the modified silver needle in a drug solution to obtain a drug-loaded acupuncture needle.

2. The method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene according to claim 1, characterized in that: The concentration of the SAC4A-SH solution in step 1 is 5 mM.

3. The method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene according to claim 1, characterized in that: The synthesis method of the SAC4A-SH is as follows: (1) Add 690 mg NH2-PEG-CSAC4A (0.57 mmol) to a 100 mL round-bottom flask and dissolve it in 40 mL phosphate buffer; (2) Add 94 mg of 2-iminothiolane hydrochloride (0.68 mmol) into a round-bottom flask and stir the mixture in the dark at room temperature under nitrogen protection for 12 hours; (3) After the reaction was completed, the product was placed in a dialysis bag with a molecular weight of 500 and dialyzed in water in the dark for 48 hours. After the dialysis was completed, the solvent was dried in a vacuum to obtain 530 mg of the product with a yield of 66%.

4. The method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene according to claim 3, characterized in that: The preparation method of the NH2-PEG-CSAC4A is as follows: (1) Add 200 mg of monocarboxytrisulfonate azocalix[4]arene (0.18 mmol) to a 100 mL round-bottom flask and dissolve it in 25 mL of N,N-dimethylformamide; (2) Add 44 mg of N,N-diisopropylethylamine (0.34 mmol) and 78 mg of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.21 mmol) into a round-bottom flask and stir at room temperature for 30 minutes; add 45 mg of tert-butyl [2-(2-aminoethoxy)ethyl]carbamate (0.22 mmol) into the above reaction system and stir at room temperature for 12 hours; vacuum spin dry the solvent to obtain a red powder; (3) The solid was added to a 100 mL round-bottom flask, dissolved with 40 mL of dimethyl sulfoxide, and then 3 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature for 6 hours. The solvent was dried under vacuum, and the product was recrystallized from water and methanol to obtain 138 mg of the product with a yield of 60%.

5. The method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene according to claim 1, characterized in that: The soaking time in step 2 is 24 to 72 hours.

6. The method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene according to claim 1, characterized in that: The method for activating the surface of the silver needle in step 2 includes: The silver needle was polished with Al2O3 suspension, ultrasonically cleaned with anhydrous ethanol and ultrapure water for 2 min each, then immersed in 6M nitric acid solution for a few seconds, mechanically scrubbed on suede, and ultrasonically cleaned with anhydrous ethanol and ultrapure water for 2 min each, with N2 protection throughout the process.

7. The method for preparing acupuncture needles with broad-spectrum quantitative drug loading based on azocalixarene according to claim 1, characterized in that: The concentration of the drug solution in step 3 is 0.5-5 mM, and the loading time each time is 10-50 min.

8. A drug-loaded acupuncture needle, characterized in that: The invention comprises a silver needle matrix and a SAC4A-SH coating loaded on the surface of the silver needle matrix. The coating is firmly combined with the surface of the silver needle through a strong interaction of thiol groups and can uniformly adsorb and release drug molecules, and is suitable for broad-spectrum drug delivery and quantitative release.

9. The drug-loaded acupuncture needle according to claim 1, characterized in that: The acupuncture needles are prepared by the following method: (1) Activate the surface of the silver needle substrate to remove oxides; (2) Soaking the activated silver needle in a SAC4A-SH solution to allow SAC4A-SH to be firmly attached to the surface of the silver needle via the thiol group; (3) Soak the modified silver needles in a drug solution to load the target drug molecules.

10. The drug-loaded acupuncture needle according to claim 1, characterized in that: The SAC4A-SH coating has a highly adjustable drug loading capacity, with an attached drug concentration of 0.5-5 mM, and has a sustained-release function, which can stably release drugs during use.

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