A SOD-containing patch for protecting bone joints and cartilage tissue

Abstract

The application discloses a SOD-containing patch for protecting bone joints and cartilage tissue and belongs to the fields of biological medicine and medical devices. The patch is in the form of a polymer microneedle, comprising a base layer and a needle tip layer; the needle tip layer comprises microneedles arranged in an array on the base layer, and the microneedles are loaded with SOD; and the base material of the microneedles is a high-molecular compound containing sodium hyaluronate and chondroitin sulfate. The patch has good anti-inflammatory, repairing and pain-relieving effects, all active ingredients in the patch are substances contained in the human body, the safety is extremely good, and the SOD is wrapped with sodium hyaluronate-chondroitin sulfate composite hydrogel, so that the SOD can be prevented from softening due to moisture absorption, thereby guaranteeing the treatment effect.

Description

Technical Field

[0001] This invention relates to an SOD-containing patch for protecting bone, joints, and cartilage tissue, belonging to the fields of biomedicine and medical devices. Background Technology

[0002] With increasing life expectancy, population aging brings new health challenges, with osteoarthritis becoming a common disease that seriously threatens the health of middle-aged and elderly people. The treatment of cartilage and osteoarthritis has always been a difficult problem, generally occurring in the middle to late stages. Meniscus removal, lumbar discectomy, and artificial joint replacement are considered the best treatments for cartilage and osteoarthritis. However, these surgeries involve large incisions, significant damage, and compatibility issues with exogenous prostheses, severely impacting the quality of life for the elderly. Furthermore, research shows that middle-aged and elderly people, white-collar workers, sports enthusiasts, overweight individuals, those living in cold and damp areas, and those engaged in housework all have lower self-reported cartilage and osteoarthritis health scores than average. Therefore, improving cartilage and osteoarthritis health through daily care is an important way to prevent osteoarthritis and has gained widespread recognition and attention both domestically and internationally.

[0003] Medical research has found that chondroitin sulfate plays a crucial role in the repair of articular cartilage. Patent CN114558176A provides sodium hyaluronate-chondroitin sulfate nanoparticles as a joint lubricant, in which the active ingredient, chondroitin sulfate, possesses cartilage repair capabilities and can promote cartilage growth. Chondroitin sulfate is one of the main components of cartilage, constituting its structure, absorbing and retaining moisture, and providing nutrients. In its 2003 recommendations for the treatment of osteoarthritis published by the European Federation for the Prevention of Rheumatism, it was considered an effective drug for treating knee osteoarthritis. The amount of chondroitin sulfate in the human body gradually decreases around the age of 20; therefore, with age, oral intake of a certain dose of chondroitin sulfate can significantly improve cartilage and joint health, playing an important role in preventing related diseases.

[0004] Superoxide dismutase (SOD) is an important antioxidant enzyme widely distributed in various organisms, such as animals, plants, and microorganisms. SOD possesses unique physiological activity, directly converting free radicals in the human body into water and oxygen, acting as the body's first line of defense against free radicals. Numerous studies have demonstrated that when cartilage and joints are damaged or diseased, a significant amount of reactive oxygen species (ROS) are produced locally, and the level of SOD in the body is significantly reduced. Increasing SOD expression in bone and cartilage, or directly injecting exogenous SOD into the affected area, can effectively achieve anti-inflammatory and repair effects. To date, there are very few reports on the use of exogenous oral SOD to improve cartilage and joint health.

[0005] Sodium hyaluronate is a mucopolysaccharide widely found in human connective tissue and synovial fluid, possessing analgesic, anti-inflammatory, and anti-damage properties. Patent CN109965104A discloses a patch containing sodium hyaluronate for joint protection, wherein the sodium hyaluronate lubricates the joints. As a joint fluid supplement, sodium hyaluronate can alleviate pain and improve joint function, and is widely used clinically.

[0006] Soluble microneedles are currently the most widely researched type of microneedle product, possessing irreplaceable advantages over other percutaneous methods, such as painlessness, minimal invasiveness, controllable drug release, and ease of use. They have enormous application and market potential in fields such as medicine. However, current microneedles for protecting bone and cartilage tissue still face challenges, including sodium hyaluronate microneedles being susceptible to moisture, affecting their mechanical strength and making them difficult to insert into the skin, and requiring stringent storage conditions. Additionally, SOD, as an enzyme, is prone to inactivation during the microneedle preparation process. Summary of the Invention

[0007] [Technical Issues]

[0008] The technical problem to be solved by the present invention is to formulate a microneedle patch by compounding SOD, chondroitin sulfate and sodium hyaluronate.

[0009] [Technical Solution]

[0010] This invention provides an SOD-containing patch for protecting bone and cartilage tissue. The patch is structured as polymer microneedles, comprising a base layer and a tip layer. The tip layer comprises microneedles (needle tips) arranged in an array on the base layer, and the microneedles are loaded with SOD. The substrate of the microneedles (needle tip layer) is a polymeric composite containing sodium hyaluronate and chondroitin sulfate.

[0011] The microneedle is conical, with a base diameter of 200 μm, a height of 750 μm, and a tip diameter of 5-10 μm.

[0012] The mass ratio of sodium hyaluronate to chondroitin sulfate in the substrate of the microneedles is 1:1-15:1, preferably 9:1-12:1.

[0013] The amount of SOD loaded in the microneedles can be 250-2000 IU / microneedle, preferably 500 IU / microneedle. The SOD is superoxide dismutase. The type of SOD is one or more of Cu / Zn-SOD, Mn-SOD, Fe-SOD, and all other subtypes. The source of SOD includes extraction from plants and animals and SOD obtained through microbial fermentation.

[0014] The base layer material is one of PE film, PVC film, PET film, EVA film, PU film, PSA film, PVA film, and PVA / PVP composite film.

[0015] This invention provides a method for preparing an SOD-containing patch for protecting bone, joints, and cartilage tissue, comprising the following steps:

[0016] (1) Dissolve PVA in sterile water to obtain dispersion system A;

[0017] (2) Sodium hyaluronate and chondroitin sulfate were added to dispersion system A and stirred to obtain hydrogel B;

[0018] (3) Add SOD to hydrogel B, stir, centrifuge, remove air bubbles, and obtain needle tip dispersion C;

[0019] (4) Pour the needle tip dispersion C into the microneedle mold and dry it;

[0020] (5) Soak the mold and patch together in a 10-30% glycerin solution for 6-12 hours;

[0021] (6) Dry the mold and the patch, cover the base layer on the surface of the mold, and peel off to obtain an SOD-containing patch for improving cartilage and joint health.

[0022] In one embodiment of the present invention, the average molecular weight Mn of PVA in step (1) is 10,000-300,000.

[0023] In one embodiment of the present invention, the mass ratio of sodium hyaluronate to chondroitin sulfate in step (2) is 1:1-15:1, preferably 9:1-12:1. The molecular weight of the sodium hyaluronate is 10,000-2,000,000, preferably 100,000-1,500,000.

[0024] In one embodiment of the present invention, the concentration of chondroitin sulfate in the needle tip dispersion C in step (2) is 2%.

[0025] In one embodiment of the present invention, the immersion time of the mold in the glycerol solution in step (5) is 8-12 hours.

[0026] In one embodiment of the present invention, the concentration of the glycerol solution in step (5) is 15-20%.

[0027] In one embodiment of the present invention, the method includes the following steps:

[0028] (1) Add 20g PVA to 50mL of sterile water and dissolve it at 70℃ to obtain dispersion system A;

[0029] (2) Weigh 12-24g of sodium hyaluronate (molecular weight 800,000-1,500,000) and 2g of chondroitin sulfate, add them to dispersion system A, and stir at 4℃ and 800rpm for 1-5h to obtain hydrogel B;

[0030] (3) Weigh 50g SOD, add it to hydrogel B, and make up to 100mL with sterile water. Stir at 1000rpm for 10min, centrifuge at 5000rpm for 5min to remove air bubbles from the solution and obtain needle tip dispersion C.

[0031] (4) Pour the needle tip dispersion C into the microneedle mold, and the liquid layer formed in the mold is about 0.2 mm thick. Dry it at 40°C for 3-4 hours.

[0032] (5) Remove the microneedle mold and immerse the patch and the mold together in a 10-30% glycerin solution for 6-12 hours;

[0033] (6) Dry at 40°C for 2 hours, remove the mold, cover the surface with a base layer, and peel off to obtain an SOD-containing patch for improving cartilage and joint health.

[0034] [Beneficial Effects]

[0035] This invention uses a specific dosage of SOD, chondroitin sulfate, and sodium hyaluronate as active ingredients in a compound formulation. Through dosage optimization and ingredient adjustment, it achieves excellent anti-inflammatory, repairing, and analgesic effects. Furthermore, since all active ingredients in the patch are substances naturally present in the human body, it has excellent safety profile and is unlikely to cause adverse reactions, showing promising application prospects in improving cartilage and joint health.

[0036] Compared to other drug delivery formulations, this invention encapsulates SOD with a sodium hyaluronate-chondroitin sulfate composite hydrogel, ensuring that SOD is not easily deactivated during preparation and storage, thereby guaranteeing therapeutic efficacy. Sodium hyaluronate and chondroitin sulfate serve not only as the substrate for patch formation but also as important active ingredients for treating osteoarthritis.

[0037] The microneedle patch of the present invention is more rigid, can prevent moisture absorption and softening, is not sensitive to environmental temperature and humidity, and can be stored at room temperature. Attached Figure Description

[0038] Figure 1 : Schematic diagram of the structure of the SOD patch in the embodiment.

[0039] Figure 2 Results of pain assessment in a rat osteoarthritis model using SOD-containing patches.

[0040] Figure 3 Results of the effect of SOD-containing patches on IL-6 levels in the synovial fluid of a rat model of osteoarthritis.

[0041] Figure 4 Results of the effect of SOD-containing patches on the level of α (TNF-α) in the synovial fluid of a rat model of osteoarthritis. Detailed Implementation

[0042] Example 1: A method for preparing and optimizing an SOD-containing patch for protecting bone, joints, and cartilage tissue.

[0043] like Figure 1 As shown, an SOD-containing patch for protecting bone, joints, and cartilage tissue includes a base layer 1 and an SOD-loaded needle tip layer 2. The soluble microneedles are arrayed on the base layer. The substrate of the base layer 1 is a PET film. The needle tip layer 2 contains SOD 21. The substrate 22 of the needle tip layer 2 is a sodium hyaluronate-chondroitin sulfate polymeric composite hydrogel. Sodium hyaluronate and chondroitin sulfate serve as both the substrate for the microneedle tip layer and important active ingredients. Each SOD-containing patch has 100 microneedles arranged in a 10×10 array in the needle tip layer 2. Each microneedle is 750 μm high, with a needle tip diameter of 5-10 μm, a needle base diameter of 200 μm, and a distance of 800 μm between needle tips. Each SOD-containing patch is circular, with an area of ​​1.5 cm². 2 .

[0044] Specifically, the sodium hyaluronate (molecular weight 800,000-1,500,000) used in this embodiment was from Shanghai Maclean Biochemical Technology Co., Ltd., and the chondroitin A sodium salt was from Shanghai Aladdin Biochemical Technology Co., Ltd. SOD was from Shenzhen Zhongke Xinyang Biotechnology Co., Ltd. The specific activity of SOD was 50,000 IU / g.

[0045] Specifically, the SOD-containing patch used to protect bone and cartilage tissue in this embodiment is prepared using the following steps:

[0046] (1) Add 20g PVA to 50mL of sterile water and dissolve it at 70℃ to obtain dispersion system A;

[0047] (2) Weigh 12-24g of sodium hyaluronate (molecular weight 800,000-1,500,000) and 2g of chondroitin sulfate, add them to dispersion system A, and stir at 4℃ and 800rpm for 1-5h to obtain hydrogel B;

[0048] (3) Weigh 50g SOD, add it to hydrogel B, and make up to 100mL with sterile water. Stir at 1000rpm for 10min, centrifuge at 5000rpm for 5min to remove air bubbles from the solution and obtain needle tip dispersion C.

[0049] (4) Pour the needle tip dispersion C into the microneedle mold, and the liquid layer formed in the mold is about 0.2 mm thick. Dry it at 40°C for 3-4 hours.

[0050] (5) Remove the microneedle mold and immerse the patch and the mold together in a 10-30% glycerin solution for 6-12 hours;

[0051] (6) Dry at 40°C for 2 hours, remove the mold, cover the surface with a base layer, and peel off to obtain an SOD-containing patch for improving cartilage and joint health.

[0052] In this embodiment, since the volume of the drug-containing microneedles in each patch is approximately 20 μL and the SOD enzyme activity is 25000 IU / ml, the drug loading of the patch should be 500 IU.

[0053] In this embodiment, the mass ratio of sodium hyaluronate to chondroitin sulfate of different molecular weights was compared and optimized using two parameters: moisture resistance and SOD enzyme activity, as well as the soaking time in step (6). During the experiment, five patches were selected from each comparative example for testing. The specific evaluation method is as follows:

[0054] (1) Moisture resistance: The prepared patch was placed in an environment with a relative humidity of 80% and a temperature of 30℃ for 0, 1, 7 and 30 days to test the mechanical properties of the microneedles.

[0055] Mechanical property testing method: Frozen rabbit skin was immersed in physiological saline to restore it to room temperature, and the surface moisture was wiped off. In a ventilated area, the prepared patch was vertically inserted into the rabbit skin and held for 2 minutes. The patch was then removed from the mucosa, and the treated rabbit skin was soaked in 10% formalin, embedded in paraffin, and sectioned longitudinally along the skin with a section thickness of 20 μm. The tissue cross-section was observed under an optical microscope, and the insertion depth was measured. In principle, the deeper the patch is inserted into the skin, the higher the mechanical strength.

[0056] (2) SOD enzyme activity: Dissolve the prepared patch in 1 mL of distilled water to obtain the test sample. The SOD enzyme activity was detected by NBT method and Beyotime reagent kit (product number: S0109). Each sample was tested three times and the average value was taken.

[0057] The specific methods for enzyme activity assay are as follows:

[0058] (a) Preparation of NBT / enzyme working solution: Prepare an appropriate amount of NBT / enzyme working solution according to a volume of 160 μL for each reaction.

[0059] Mix 158 μL of SOD detection buffer, 1 μL of NBT, and 1 μL of enzyme solution evenly to prepare a 160 μL NBT / enzyme working solution. Prepare an appropriate amount of NBT / enzyme working solution according to the number of samples to be tested (including standards).

[0060] (b) Preparation of the reaction initiation working solution: Dissolve and mix the reaction initiation solution (40X) from the kit (Beyotime, product number: S0109). Dilute the solution by adding 39 μL of SOD detection buffer to every 1 μL of reaction initiation solution (40X). Mix well to obtain the reaction initiation working solution. Prepare an appropriate amount of reaction initiation working solution according to the number of samples to be tested (including standards). Store the prepared reaction initiation working solution at 4°C or on ice. It can be used on the same day, but it is recommended to prepare and use it immediately.

[0061] (c) Sample determination: Refer to the table below to set up sample wells and various blank control wells using a 96-well plate. Add the test samples and other solutions in the order listed in the table below. After adding the reaction start-up working solution, mix thoroughly. React at 37°C for 30 min, and measure the absorbance at 560 nm.

[0062] Table 1. Preparation of SOD enzyme activation solution

[0063]

[0064] Calculation of total SOD activity in the test sample: Inhibition percentage = [(A blank control 1 - A blank control 2) - (A test sample - A blank control 3)] / (A blank control 1 - A blank control 2) × 100%

[0065] In the detection system, the SOD enzyme activity unit = inhibition percentage / (1 - inhibition percentage) units.

[0066] The results are shown in Table 2 below. Comparative Examples 1-5 mainly optimized the effect of the mass ratio of sodium hyaluronate to chondroitin sulfate (with the concentration of chondroitin sulfate in dispersion C fixed at 2% (g / 100mL)) on the moisture resistance and SOD activity of the patch. The results showed that the microneedles prepared with a mass ratio of sodium hyaluronate to chondroitin sulfate of 9:1-12:1 exhibited the best mechanical properties and SOD activity. In comparison Examples 1 and 6-9, the main optimization step (6) involved soaking the mold in glycerin for 8-1... The patch obtained after 2 hours has the best overall performance; in Comparative Examples 1 and 10-12, the main optimization step (6) is the concentration of glycerin. When the glycerin concentration is 15-20%, the patch has the best mechanical properties and SOD enzyme activity; after comprehensive comparison, the patch has the best overall performance, including moisture resistance and SOD enzyme activity, when the mass ratio of sodium hyaluronate to chondroitin sulfate is 9:1-12:1, the mold soaking in glycerin in step (6) is 8-12 hours, and the concentration of glycerin in step (6) is 15-20%.

[0067] Table 2 shows the optimized manufacturing method of the patch.

[0068]

[0069] Example 2: Therapeutic effect of SOD-containing patch on rat osteoarthritis model

[0070] This embodiment optimizes and compares the therapeutic effects of SOD patches on osteoarthritis in vivo by optimizing SOD content and substrate composition, as detailed below:

[0071] 1. How to make SOD patches:

[0072] (1) Following the preparation method of Example 1, and fixing the concentration of chondroitin sulfate in needle tip dispersion C to 2%, the concentration of sodium hyaluronate in needle tip dispersion C to 18% (mass ratio of sodium hyaluronate to chondroitin sulfate 9:1), the glycerol concentration in step (5) to 20%, and the soaking time to 10h, SOD patches with different SOD contents were prepared, namely Comparative Example 1 and Comparative Examples 16-19.

[0073] (2) Referring to the preparation method of Example 1, the concentration of sodium hyaluronate in the needle tip dispersion C was fixed at 18%, and the chondroitin sulfate in step (2) of Example 1 was replaced with the same mass of polyvinyl alcohol (PVA). The glycerol concentration in step (5) was 20%, the soaking time was 10h, and the other steps were the same. SOD sodium hyaluronate-polyvinyl alcohol microneedles were prepared, namely Comparative Example 13.

[0074] (3) Referring to the preparation method of Example 1, the concentration of chondroitin sulfate in the needle tip dispersion C was fixed at 2%, and the sodium hyaluronate in step (2) of Example 1 was replaced with the same mass of polyvinyl alcohol (PVA). The glycerol concentration in step (5) was 20%, the soaking time was 10h, and the other steps were the same. SOD chondroitin sulfate-polyvinyl alcohol microneedles were prepared, namely Comparative Example 14.

[0075] (4) Referring to the preparation method of Example 1, the chondroitin sulfate in step (2) of Example 1 was replaced with the same mass of polyvinyl alcohol (PVA), and the sodium hyaluronate was also replaced with the same mass of polyvinyl alcohol (PVA). The glycerol concentration in step (5) was 20%, the soaking time was 10h, and the other steps were the same. SOD polyvinyl alcohol microneedles were prepared, namely Comparative Example 15.

[0076] (5) Following the preparation method in steps (1)-(3) of Example 1, the needle tip dispersion C was obtained. The needle tip dispersion C was then directly poured into a container with an area of ​​1.5 cm². 2 A hydrogel patch, namely Comparative Example 20, is prepared by drying a liquid layer with a thickness of about 0.2 mm in a circular planar groove at 40°C for 1.5-2 hours.

[0077] 2. Methods for establishing an osteoarthritis model: SPF-grade SD rats, healthy males, 6-8 weeks old, weighing 180g-210g, were randomly divided into 11 groups of 10 rats each: normal control group, model group, control group 1, and groups 13-20. Before the experiment, the rats were fasted for 12 hours and anesthetized by intraperitoneal injection of 10% chloral hydrate (0.3mL / 100g). After anesthesia, the rats were fixed on the operating table, and the hair on the left hind knee joint was shaved off. The area was disinfected with iodine. The rat knee joint was slightly flexed, and the syringe was inserted obliquely upwards and downwards from the medial side of the patella into the joint cavity. The control group was injected with 50μL of physiological saline, while the other groups were injected with 50μL of sodium iodoacetate (60mg / mL). After injection, the rats were placed in animal cages for free movement and feeding.

[0078] 3. Administration method: Administer the medication starting the day after surgery, once every two days, one patch (dose) each time, for a total of 14 administrations. The normal control group consisted of normal rats that did not undergo modeling and received no medication; the model group consisted of arthritis rats that underwent modeling but did not receive medication.

[0079] 4. Evaluation indicators:

[0080] (1) Pain assessment during the procedure was performed as follows: The rat's head was fixed, and its hind legs and tail were extended outside the fixation tube. After 5 minutes to allow the rat to adapt to the environment, the measurement was performed. The rat's joints were slowly and gently bent, once every 5 seconds, for a total of 5 times. If the rat showed a short and obvious leg retraction or squealing response, the score was 1 point; if there was no response, the score was 0. The scores of leg retraction and squealing were used to reflect the degree of joint pain. The assessment was performed before the operation (recorded as day 0), and on days 1, 14, and 28 after drug administration.

[0081] (2) Detection method for interleukin (IL-6) and TNF-α levels in rat joint lavage fluid:

[0082] The joint cavity of each rat was irrigated with 100 μL of PBS. The irrigated fluid was collected, centrifuged at 3500 rpm for 15 min, and the supernatant was aliquoted and administered via ELISA (Mouse Interleukin (IL-6) ELISA Kit (Shanghai Yuanmu Biotechnology Co., Ltd.)) and Xinbosheng within one week. The ELISA kit was used to detect the levels of interleukin (IL-6) and TNF-α.

[0083] Table 3. Components and SOD content used in step (2) of each comparative example.

[0084]

[0085] result:

[0086] (1) As Figure 2 As shown: At day 14, the model group had the highest pain index, while the pain indices of the other treatment groups were lower than those of the model group. By day 28, the pain indices of all groups decreased. Comparison of Comparative Example 1 and Comparative Example 20 indicates that microneedle delivery has better transdermal efficacy for large molecules such as SOD compared to traditional patch delivery. Comparison of the model group and Comparative Examples 1, 16, 17, 18, and 19 shows that 250 IU-2000 IU SOD patches have a significant effect in relieving osteoarthritis, with the effect becoming more pronounced with higher doses. On the other hand, comparison of optimized Comparative Example 15 with Comparative Examples 1, 13, and 14 reveals that Comparative Examples 1, 13, and 14 show a significant decrease in pain index, with Comparative Example 1 showing the best effect. This indicates that sodium hyaluronate and chondroitin sulfate, when combined with SOD, serve not only as the substrate for patch formation in this invention but also as important active ingredients for treating osteoarthritis.

[0087] (2) Figure 3-4As shown, by comparing the normal control group and comparative examples 1, 16-19, it was shown that 250 IU-2000 IU SOD patches had a significant inhibitory effect on the expression of inflammatory factors interleukin IL-6 and tumor necrosis factor TNF-α in synovial fluid. SOD patches of 500 IU and above could keep the levels of IL-6 and TNF-α in model rats between normal levels, with no significant difference from the normal control group, and played a good protective role for cartilage and joints.

Claims

1. An SOD-containing patch for protecting bone, joints, and cartilage tissue, characterized in that, The structure is a polymer microneedle, comprising a base layer and a tip layer; the microneedles are conical, with a base diameter of 200 μm, a height of 750 μm, and a tip diameter of 5-10 μm; the tip layer comprises microneedles arranged in an array on the base layer, and the microneedles are loaded with SOD; the substrate of the microneedles is a polymeric composite containing sodium hyaluronate and chondroitin sulfate, and the preparation method includes the following steps: (1) Dissolve 20 g of PVA in 50 mL of sterile water at 70 °C to obtain dispersion system A; the average molecular weight Mn of PVA is 10,000-300,000; (2) Add 18-24 g of sodium hyaluronate and 2 g of chondroitin sulfate to dispersion system A, and stir at 4℃ and 800 rpm for 1-5 h to obtain hydrogel B; the molecular weight of sodium hyaluronate is 800,000-1,500,000. (3) Add 50g SOD to hydrogel B, make up to 100 mL with sterile water, stir at 1000 rpm for 10 min, centrifuge at 5000 rpm for 5 min to remove air bubbles, and obtain needle tip dispersion C. (4) Pour the needle tip dispersion C into the microneedle mold, and the liquid layer formed in the mold is 0.2 mm thick. Dry it at 40°C for 3-4 h. (5) Remove the microneedle mold and immerse the mold and patch together in a 15-20% glycerin solution for 8-12 hours; (6) Dry the mold and patch at 40°C for 2 hours, cover the base layer on the surface of the mold, and peel off to obtain a patch containing SOD for improving cartilage and joint health; the base layer material is one of PE film, PVC film, PET film, EVA film, PU film, PSA film, PVA film and PVA / PVP composite film.

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