Use of a piezoelectric material in the preparation of a medicament for the treatment of gout

Abstract

The application relates to application of a piezoelectric material in preparation of a medicine for treating gout, characterized in that a medicine active ingredient of the medicine comprises the piezoelectric material, the piezoelectric material is barium titanate; the application comprises the following steps: delivering the medicine to a lesion site, applying ultrasonic stimulation to the lesion site, and exciting in-vivo active species degradation of uric acid by the medicine. The piezoelectric material has good piezoelectric catalytic performance, excellent tissue penetration ability, low toxicity and high precision, active species can be controllably excited by means of ultrasonic action at the time of gout attack, uric acid can be efficiently degraded into allantoin which is good in solubility and non-toxic, symptoms can be rapidly relieved, the piezoelectric material can be long-term resident in joints, can keep stable catalytic effect in multiple cycles, and long-term treatment of gout can be realized by single injection, and a new gout treatment scheme which is more effective and more durable is provided.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of a piezoelectric material in the preparation of drugs for treating gout. Background Technology

[0002] Gout is a common inflammatory disease related to uric acid disorders. Its main cause is the deposition of urate crystals in the joints, clinically manifesting as sudden, severe joint swelling and pain, and recurrent attacks. In severe cases, it can lead to structural joint damage. In my country, gout has become the second most common metabolic disease after diabetes, and its incidence is increasing year by year and affecting younger people, posing a significant public health threat to the health of Chinese residents. Currently, the main clinical methods for relieving and treating gout are various oral uric acid-lowering drugs, such as febuxostat, allopurinol, and benzbromarone. In recent years, enzyme catalytic therapy, using bioenzymes or nanoenzymes with catalytic properties to achieve rapid degradation of uric acid, has gradually become a research hotspot in the field of gout treatment. Uricase is an important bioenzyme that can convert uric acid into allantoin, which is highly soluble and non-toxic, thus relieving gout symptoms. Researchers have explored many schemes to optimize the performance and delivery efficiency of uricase, focusing on improving its therapeutic effect through enzyme recombination, chemical modification, or improvements to drug delivery systems. In addition, the design and development of novel nanozymes with high efficiency and stability are also being actively promoted. By simulating the active center structure of natural uricase, novel uricase replacement systems with high catalytic activity and good stability are being developed.

[0003] However, current oral uric acid-lowering drugs used for gout relief and treatment may cause liver and kidney damage or pose potential cardiovascular risks, and long-term oral administration places high demands on patient adherence. Furthermore, gout patients have a high probability of experiencing recurrent attacks, and symptoms tend to worsen with increasing frequency of attacks. Natural uricase extracted from organisms has limitations such as high immunogenicity, high production costs, and poor stability, restricting its further widespread application. In addition, gout is difficult to cure and prone to frequent attacks, making long-term relief and treatment a challenging problem. Currently, neither biological enzymes nor nanozymes have achieved sustained long-term therapeutic effects after a single dose. Various enzymes reported so far spontaneously activate their degradation behavior of uric acid when near it, unaffected by uric acid concentration. This uncontrolled degradation makes it difficult for enzymes to exert their effects for extended periods, meaning patients will need to take medication again when gout recurs, undoubtedly increasing the difficulty and cost of disease management. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of piezoelectric materials in the preparation of drugs for treating gout, characterized in that the active pharmaceutical ingredient of the drug includes the piezoelectric material, wherein the piezoelectric material is barium titanate (BaTiO3). The application includes: delivering the drug to the lesion site, applying ultrasound stimulation (US) to the lesion site, and the drug stimulating the degradation of uric acid by active species in the body.

[0005] In one embodiment, the in vivo active species include: hydroxyl radicals (·OH) and superoxide radicals (·O). 2- At least one of the following.

[0006] In one embodiment, the piezoelectric material in the drug has a mass concentration of 0.01~10 mg / mL.

[0007] In one embodiment, the delivery method includes local injection.

[0008] In one embodiment, the conditions for the ultrasound stimulation include: frequency: 1 kHz to 10 MHz; duty cycle: 1% to 100%; intensity: 0.01 to 10 W / cm². 2 Time: 0.1~1000 minutes.

[0009] In one embodiment, the conditions for the ultrasound stimulation include: frequency: 1MHz~10MHz; duty cycle: 10%~50%; intensity: 1~10W / cm². 2 Time: 10~100 minutes.

[0010] A second aspect of the present invention also provides a method for preparing the piezoelectric material in the above-mentioned applications, comprising the following steps: Synthesis of sodium tritiate (Na2Ti3O7): Titanium dioxide (TiO2) was dispersed in sodium hydroxide (NaOH) solution, and a suspension was obtained by alternating stirring and sonication. The suspension was subjected to hydrothermal reaction, and the precipitate was collected by centrifugation and washed to obtain sodium tritiate. Synthesis of layered titanate (H2Ti3O7) nanotubes: Sodium tritiate was dispersed in hydrochloric acid solution, stirred, centrifuged to collect the precipitate, washed, and dried to obtain layered titanate nanotubes. Synthesis of piezoelectric materials: Barium hydroxide octahydrate (Ba(OH)2·8H2O) is dispersed in water with nitrogen (N2) continuously introduced, and the mixture is stirred and sonicated alternately. After stopping the nitrogen supply, layered titanate nanotubes are added by stirring and sonicating alternately. The mixture undergoes a hydrothermal reaction, and the precipitate is collected by centrifugation, washed, and dried to obtain the piezoelectric material.

[0011] In one embodiment, the ratio of titanium dioxide to sodium hydroxide solution to hydrochloric acid solution is (0.01~10) g : (0.01~100) mL : (0.01~350) mL, the concentration of sodium hydroxide solution is 10 mol / L, and the concentration of hydrochloric acid solution is 1 mol / L. According to the mass ratio, the octahydrate barium hydroxide : the layered titanate nanotubes are (0.01~10):1.

[0012] In one embodiment, the titanium dioxide : sodium hydroxide solution : hydrochloric acid solution is (0.5~5) g : (50~100) mL : (200~350) mL in the dosage ratio, the concentration of the sodium hydroxide solution is 10 mol / L, and the concentration of the hydrochloric acid solution is 1 mol / L. According to the mass ratio, the octahydrate barium hydroxide : the layered titanate nanotubes are (5~10):1.

[0013] In one embodiment, in the synthesis of sodium trititanate, the hydrothermal reaction is carried out at a temperature of 120~300°C for a time of 1~120h. In the synthesis of the piezoelectric material, the hydrothermal reaction temperature is 120~300℃ and the time is 1~120h.

[0014] In one embodiment, in the synthesis of sodium trititanate, the hydrothermal reaction is carried out at a temperature of 200-300°C for a time of 20-80 hours. In the synthesis of the piezoelectric material, the hydrothermal reaction temperature is 200~300℃ and the time is 20~80h.

[0015] In one embodiment, the alternating stirring and sonication in the synthesis of sodium tritiate consists of alternating stirring for 1-100 min and sonication for 1-100 min.

[0016] In one embodiment, the centrifugation time in the synthesis of sodium trititanate is 1 to 10 minutes.

[0017] In one embodiment, during the synthesis of the sodium trititanate, the washing is performed with a hydrochloric acid solution at a concentration of 1 mol / L.

[0018] In one embodiment, the stirring time during the synthesis of the layered titanate nanotubes is 1 to 20 hours.

[0019] In one embodiment, during the synthesis of the layered titanate nanotubes, the stirring is performed by stirring for a certain period of time, stopping the stirring, sonicating for 5-60 minutes, and then continuing to stir.

[0020] In one embodiment, the centrifugation time in the synthesis of the layered titanate nanotubes is 1 to 10 minutes.

[0021] In one embodiment, during the synthesis of the piezoelectric material, the alternating stirring and ultrasonication involves alternating stirring for 1-100 minutes and ultrasonication for 1-100 minutes.

[0022] In one embodiment, the centrifugation time during the synthesis of the piezoelectric material is 1 to 10 minutes.

[0023] In one embodiment, during the synthesis of the piezoelectric material, the washing process involves first washing with 0.2 mol / L hydrochloric acid solution, then washing with water, and finally washing with anhydrous ethanol.

[0024] In one embodiment, the drying time in the synthesis of the piezoelectric material is 2-5 hours.

[0025] A third aspect of the present invention also provides a medicament for treating gout, comprising a pharmaceutically active ingredient and pharmaceutically acceptable excipients, wherein the pharmaceutically active ingredient comprises a piezoelectric material used in the above applications or a piezoelectric material obtained by the above preparation method.

[0026] In one embodiment, the excipients include at least one of excipients, lubricants, antioxidants, preservatives, binders, fillers, or thickeners.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. The materials used in this invention are prepared by myself, the method is simple, inexpensive, safe and reliable.

[0028] 2. This invention reverses the physiological mechanism by which uric acid can clear active species. The selected piezoelectric catalytic material has excellent piezoelectric catalytic performance, which can efficiently generate active species and degrade uric acid into soluble and non-toxic allantoin. The catalytic performance is stable, and it also has the characteristics of strong tissue penetration, low toxicity, good biocompatibility and high precision.

[0029] 3. The piezoelectric catalytic gout treatment system constructed in this invention can be precisely controlled to start and stop the piezoelectric catalytic reaction in real time with the help of an external ultrasound source. Its excellent performance allows it to repeatedly generate active species to degrade uric acid, and can achieve a long-lasting and controllable uric acid degradation effect in gout treatment with a single injection. Attached Figure Description

[0030] Figure 1 The X-ray diffraction pattern of the prepared BaTiO3 nanoparticles; Figure 2 Scanning electron microscope image of the prepared BaTiO3 nanoparticles; Figure 3 The piezoelectric catalytic diagram of the prepared BaTiO3 nanoparticles is shown, where A is the change of uric acid concentration in each group within 80 minutes, and B is the rate constant of uric acid degradation reaction in each group. Figure 4 Figure 1 shows the cytotoxicity of BaTiO3 nanoparticles at different concentrations. Figure 5 Fluorescence images of IL-1β, TNF-α, iNOS and NLRP3 in Raw 264.7 cells cultured under different conditions; Figure 6 The protein expression of IL-1β, TNF-α, and INOS in Raw264.7 cells cultured under different conditions was determined by Western blotting. Detailed Implementation

[0031] The technical solution of this invention utilizes barium titanate (BaTiO3), a piezoelectric material capable of in-situ, repeatable, and controllable degradation of uric acid through a single injection. It exhibits excellent piezoelectric catalytic performance, outstanding tissue penetration, low toxicity, and high precision. During a gout attack, it can controllably activate active species (·O) using ultrasound (US). 2- BaTiO3 (containing substances such as ·OH) efficiently degrades uric acid into allantoin, a highly soluble and non-toxic compound, rapidly relieving symptoms. Furthermore, BaTiO3 is chemically stable, can remain in the joint for a long time, and maintains a stable catalytic effect through multiple cycles, achieving long-term gout treatment with a single injection. Based on piezoelectric catalysis, this novel gout treatment approach utilizes piezoelectric catalytic materials with highly efficient, controllable, and long-lasting effects, providing a more effective and durable solution.

[0032] In the preparation of the piezoelectric material of this invention, which enables in-situ reproducible and controllable degradation of uric acid with a single injection, titanium dioxide (TiO2) is first dispersed in an aqueous solution of sodium hydroxide (NaOH) to synthesize layered titanate (H2Ti3O7) nanotubes via a hydrothermal reaction. These nanotubes are then dispersed in an aqueous solution of barium hydroxide octahydrate (Ba(OH)2·8H2O) and prepared again via a hydrothermal reaction to obtain BaTiO3 nanoparticles. The piezoelectric material BaTiO3 nanoparticles prepared by this invention can convert mechanical energy into electrical energy under the action of ultrasound, thereby activating active species. Based on the physiological characteristics of uric acid—namely, its ability to scavenge active species to enhance cellular antioxidant levels and its own physiological properties of being decomposed into highly soluble allantoin—this invention efficiently degrades uric acid into highly soluble and non-toxic allantoin, promptly relieving gout symptoms. Furthermore, with the assistance of an external ultrasound source, the BaTiO3 nanoparticles can precisely achieve the instantaneous initiation and termination of the catalytic reaction, enabling controllable degradation of uric acid. In addition, the excellent reusability and stable chemical properties of BaTiO3 nanoparticles allow them to remain in the joint cavity for a long time after a single injection, and the ultrasound source can be adjusted as needed according to the patient's condition to continuously degrade uric acid, thereby achieving long-term treatment of gout.

[0033] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0035] Unless otherwise specified, all reagents, materials, and equipment used in this embodiment are commercially available; unless otherwise specified, all test methods are conventional test methods in this field.

[0036] Example I. Preparation of Piezoelectric Materials 1. Synthesis of Na2Ti3O7 nanowires Accurately weigh 1.6 g of titanium dioxide (TiO2) powder and uniformly disperse it in 70 mL of a 10 mol / L sodium hydroxide (NaOH) solution. A homogeneous suspension is obtained by alternating magnetic stirring (600 rpm for 5 minutes) and ultrasonic treatment (3 minutes). The suspension is then transferred to a 100 mL polytetrafluoroethylene (PTFE) liner, which is placed inside a stainless steel high-pressure reactor. The reactor is then placed in a drying oven at 240°C for 24 hours. After the reaction, the reactor is allowed to cool naturally to room temperature. The reactor is then opened, and the reaction mixture in the liner is poured out. The resulting white precipitate is collected by centrifugation (3000 rpm for 3 minutes). The precipitate is then washed four times with a 1 mol / L hydrochloric acid solution until the pH of the washing solution is acidic (pH < 7.0) to thoroughly remove residual NaOH and other soluble impurities. After the final wash, the supernatant is discarded, yielding the white precipitate.

[0037] 2. Synthesis of H2Ti3O7 nanotubes The white precipitate obtained in the previous step was dispersed using a 1 mol / L hydrochloric acid solution, and the dispersed suspension was transferred to an 800 mL beaker. Hydrochloric acid solution of the above concentration was added to a final volume of 300 mL. The mixture was then placed on a magnetic stirrer and stirred continuously at 600 rpm for 4 hours to allow the Na+ to dissolve. + Ions are H + Complete displacement was performed, with stirring for 2 hours followed by stopping the stirring and sonicating for 30 minutes, then continuing magnetic stirring for another 2 hours. After stirring, the precipitate was collected by centrifugation (8000 rpm, 3 minutes). The precipitate was repeatedly washed with a large amount of deionized water until the washing solution was neutral (pH≈7.0) to remove residual acid and sodium ions. After the last wash, the supernatant was discarded, and the white precipitate was dispersed with a small amount of anhydrous ethanol. After centrifugation, the supernatant was removed, and the precipitate was transferred to a beaker and dried at 80°C for 12 hours. After the solid was completely dry, the white solid was collected with a spatula to obtain white powdered H2Ti3O7 nanotubes.

[0038] 3. Synthesis of BaTiO3 (BTO) nanoparticles Take a beaker and weigh out 70 mL of deionized water. Continuously purge the liquid with high-purity nitrogen (N2) to remove dissolved carbon dioxide and prevent the formation of barium carbonate. Simultaneously, add 4.416 g of barium hydroxide octahydrate (Ba(OH)2·8H2O). Use alternating magnetic stirring (600 rpm for 5 minutes) and ultrasonic treatment (3 minutes) to obtain a uniform suspension. Once the dispersion is uniform, stop the gas purging. Then, accurately weigh out 0.51 g of the H2Ti3O7 nanotubes synthesized in the previous step as the titanium source. Use alternating magnetic stirring (600 rpm for 5 minutes) and ultrasonic treatment (3 minutes) to uniformly disperse the H2Ti3O7 nanotubes in the barium hydroxide aqueous solution. Transfer the resulting mixed suspension to a 100 mL polytetrafluoroethylene (PTFE) liner. Place the liner into a stainless steel high-pressure reactor and then in a forced-air drying oven. The reaction was carried out at a constant temperature of 40°C for 12 hours. After the reaction was completed, the autoclave was allowed to cool naturally to room temperature. The reaction mixture in the liner was then poured out and the resulting white precipitate was collected by centrifugation (8000 rpm, 3 minutes). The precipitate was then washed four times with 0.2 mol / L hydrochloric acid solution until the washing solution was acidic (pH < 7) to completely remove any residual alkaline solution. The precipitate was then washed four times with deionized water until the pH of the supernatant was neutral. Finally, the precipitate was washed 1-2 times with anhydrous ethanol to facilitate subsequent drying. After the last wash, the supernatant was discarded, and the white precipitate was dispersed with a small amount of anhydrous ethanol. The dispersed suspension was then transferred to a weighing boat and dried in a forced-air oven at 65°C for 2-5 hours. After the solid was completely dried, the white solid was collected with a spatula to obtain white powdery barium titanate (BaTiO3, BTO) nanoparticles.

[0039] II. Treatment Methods 1. Medication preparation Dissolve 1-1000 mg of BaTiO3 nanoparticles in 100 mL of physiological saline, then disperse them thoroughly by sonication, and finally draw up the drug preparation using a sterile 1 mL insulin syringe.

[0040] 2. Injection procedure The operator gently stabilizes the mouse's ankle joint with one hand and holds the syringe in the other hand. The needle is inserted into the joint cavity (usually in the depression on the outside of the ankle joint) and slowly advanced until a feeling of emptiness is felt. After aspiration, if no blood is found, the medication is slowly and evenly injected into the joint cavity. After the injection is completed, the needle is quickly withdrawn and the needle hole is gently pressed with a sterile cotton swab for a moment to prevent the medication from seeping out.

[0041] 3. Parameter settings Set the parameters of the ultrasound therapy device (frequency: 1KHz~10MHz; duty cycle 1%~100%; intensity: 0.01~10W / cm). 2Time: 0.01 minutes to 1000 minutes.

[0042] 4. Ultrasound therapy One hour after the intra-articular injection, the affected ankle joint area was disinfected. Sufficient ultrasound coupling agent was evenly applied to the surface of the ultrasound probe and the skin of the mouse's ankle joint. Then, the ultrasound probe was gently placed on the swollen area of ​​the mouse's ankle joint and slowly rotated in a spiral motion in the joint area to ensure that the ultrasound waves were evenly stimulated throughout the entire area. The ultrasound treatment was stopped after 0.01 to 1000 minutes.

[0043] 5. After the ultrasound stimulation is completed, gently wipe away the residual coupling agent on the mouse skin with sterile gauze, and then observe the pain.

[0044] Example 1 The ultrasonic therapy device in this embodiment includes the following adjustable treatment parameters: frequency: 1MHz; duty cycle: 50%; intensity: 1.0W / cm². 2 .

[0045] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0046] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 50%; intensity: 1.0W / cm). 2 ).

[0047] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0048] Example 2 The ultrasound therapy device in this embodiment includes the following adjustable treatment parameters: frequency: 1MHz; duty cycle: 50%; intensity: 0.5W / cm². 2 .

[0049] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0050] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 50%; intensity: 0.5W / cm). 2 ).

[0051] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0052] Example 3 The ultrasound therapy device in this embodiment includes the following adjustable treatment parameters: frequency: 3MHz; duty cycle: 10%; intensity: 0.5W / cm². 2 .

[0053] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0054] 2. Set the parameters of the ultrasound therapy device (frequency: 3MHz; duty cycle: 10%; intensity: 0.5W / cm). 2 ).

[0055] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0056] Example 4 The ultrasound therapy device in this embodiment includes the following adjustable treatment parameters: frequency: 3MHz; duty cycle: 20%; intensity: 0.5W / cm². 2 .

[0057] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0058] 2. Set the parameters of the ultrasound therapy device (frequency: 3MHz; duty cycle: 20%; intensity: 0.5W / cm). 2 ).

[0059] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0060] Example 5 The ultrasound therapy device in this embodiment includes the following adjustable treatment parameters: 3MHz; duty cycle: 50%; intensity: 0.5W / cm². 2 .

[0061] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0062] 2. Set the parameters of the ultrasound therapy device (frequency: 3MHz; duty cycle: 50%; intensity: 0.5W / cm). 2 ).

[0063] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0064] Example 6 The treatment time in this embodiment includes the following adjustable treatment parameters: treatment time 5 minutes.

[0065] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0066] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 10%; intensity: 0.5W / cm). 2 ).

[0067] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle area. The ultrasound treatment can be stopped after 5 minutes.

[0068] Example 7 The treatment time in this embodiment includes the following adjustable treatment parameters: treatment time 30 minutes.

[0069] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0070] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 10%; intensity: 0.5W / cm). 2 ).

[0071] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 30 minutes.

[0072] Example 8 In this embodiment, the drug concentration includes the following therapeutic parameters: the drug concentration is 1 mg / mL.

[0073] The treatment includes the following steps: 1. Drug preparation: Dissolve 100mg of BaTiO3 nanoparticles in 100mL of physiological saline.

[0074] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 10%; intensity: 0.5W / cm). 2 ).

[0075] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0076] Example 9 In this embodiment, the drug concentration includes the following therapeutic parameters: the drug concentration is 5 mg / mL.

[0077] The treatment includes the following steps: 1. Drug preparation: Dissolve 500mg of BaTiO3 nanoparticles in 100mL of physiological saline.

[0078] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 10%; intensity: 0.5W / cm). 2 ).

[0079] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0080] Example 10 In this embodiment, the drug concentration includes the following therapeutic parameters: the drug concentration is 10 mg / mL.

[0081] The treatment includes the following steps: 1. Drug preparation: Dissolve 1000mg BaTiO3 nanoparticles in 100mL of physiological saline.

[0082] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 10%; intensity: 0.5W / cm). 2 ).

[0083] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0084] Example 11 The ultrasonic therapy device in this embodiment includes the following adjustable treatment parameters: frequency: 1MHz; duty cycle: 10%; intensity: 1.0W / cm². 2 .

[0085] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0086] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 10%; intensity: 1.0W / cm). 2 ).

[0087] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0088] Example 12 The ultrasonic therapy device in this embodiment includes the following adjustable treatment parameters: frequency: 1MHz; duty cycle: 10%; intensity: 3.0W / cm². 2 .

[0089] The treatment includes the following steps: 1. Drug preparation: Dissolve 20 mg of BaTiO3 nanoparticles in 100 mL of physiological saline.

[0090] 2. Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 10%; intensity: 3.0W / cm). 2 ).

[0091] 3. One hour after the intra-articular injection, perform ultrasound treatment on the affected ankle joint area. The ultrasound treatment can be stopped after 15 minutes.

[0092] Implementation effect evaluation 1. X-ray diffraction (XRD) The prepared BaTiO3 nanoparticles were uniformly spread onto the sample stage and then flattened. The sample stage was fixed on the X-ray diffractometer stage with a voltage of 40 kV, a current of 40 mA, and scanning angles of 5°~80° and 44°~46°, respectively, to observe the diffraction peaks of the BaTiO3 nanomaterials.

[0093] like Figure 1 X-ray diffraction data showed that the sample diffraction peaks corresponded to the standard characteristic peaks of BaTiO3. In particular, the asymmetric peak at the 45.2-degree position shown in the small box can be attributed to the overlap of the (002) and (200) crystal planes, thus confirming the tetragonal crystal phase structure of BaTiO3.

[0094] 2. Scanning electron microscope (SEM) The prepared BaTiO3 nanoparticles were fully dispersed in alcohol, spread on conductive adhesive, and dried thoroughly. After sputtering gold onto the sample surface for 30 seconds, the surface morphology of the sample was observed using a scanning electron microscope.

[0095] like Figure 2 Scanning electron microscopy images show that the prepared BaTiO3 exhibits a spherical morphology with a size of approximately 200 nm.

[0096] 3. In vitro uric acid degradation test Two mg of the prepared BaTiO3 sample was dispersed in 10 mL of a 100 µM uric acid solution. The mixture was stirred continuously for 30 minutes to reach adsorption equilibrium, and then subjected to piezoelectric catalysis in an ultrasonic cleaner. The total piezoelectric catalysis time was 80 minutes, with 1 mL of suspension sampled after every 20 minutes of ultrasonic vibration. After centrifugation to remove the catalyst, the absorbance of the sample solution at 290 nm was measured using a microplate reader to calculate the uric acid degradation rate.

[0097] from Figure 3As can be seen from the piezoelectric catalytic degradation performance test of BaTiO3 under ultrasonic irradiation: Combining the uric acid concentration change curves summarized in (A) and the degradation rate values ​​calculated in (B), it can be seen that the degradation of uric acid is almost negligible when only ultrasound or only BaTiO3 is used. However, BaTiO3 can degrade more than 80% of uric acid within 80 minutes under ultrasonic irradiation, that is, when the piezoelectric effect is triggered, uric acid will be significantly degraded.

[0098] 4. In vitro biocompatibility evaluation In a clean bench, cultured P3 generation chondrocytes were seeded into 96-well plates at a density of approximately 8000 cells / well and cultured in a cell culture incubator for about 12 hours until cell adhesion. An appropriate amount of synthetic BaTiO3 powder was weighed in advance and sterilized by irradiation with UV light in a clean bench for about two hours. BaTiO3 concentration gradients of 0.025, 0.05, 0.1, 0.15, 0.20, 0.25, and 0.3 mg / mL were prepared using DMEM complete medium. The old medium was removed from the 96-well plates, and three sub-wells were set up for each concentration. The pre-prepared BaTiO3 concentration gradients were added to each sub-well. Add 100 μL of the culture medium and place the 96-well plate in a cell culture incubator for 24 hours. After reaching the predetermined time point, remove the 96-well plate, discard the old culture medium, and then use a pipette to extract 100 μL of the pre-prepared complete culture medium containing 10% CCK-8 reagent and add it to the well plate. Place the 96-well plate in an incubator at 37°C for about 3 hours, and then remove it and place it in a microplate reader. According to the operating instructions, measure the absorbance value of each well and each concentration at a wavelength of 450 nm.

[0099] from Figure 4 It can be seen that when the concentration of BaTiO3 reaches 0.2 mg / mL, the survival rate of 3T3 cells is still as high as 80%, indicating that it has no significant cytotoxicity at this concentration. This concentration is the same as the material concentration used in the preliminary experiments such as uric acid degradation.

[0100] 5. In vitro efficacy of BaTiO3 in relieving gout I. In vitro gout inflammatory cell experiments The occurrence of gout inflammation is closely related to macrophages. When macrophages encounter excessive uric acid crystal deposits, they secrete a series of inflammatory factors, leading to a chain reaction and triggering severe acute gout inflammation. Sodium urate can induce cellular inflammatory responses. This invention investigates the therapeutic effects of barium titanate nanoparticles on barium titanate by preparing sodium urate solutions treated under different experimental conditions and co-culturing them with Raw264.7 cells.

[0101] (1) Establishing cell models and grouping them in vitro In in vitro experiments, we extracted P3 generation Raw264.7 cells and seeded them in 12-well plates at a cell seeding density of approximately 15 × 10⁶ cells / well. 4 Cells / wells were used to group experiments into: Sham group (blank control): wells were filled with complete culture medium only, without any intervention reagents; Sodium urate + PBS group (PBS group): wells were filled with complete culture medium containing sodium urate only; Sodium urate + BaTiO3 group (BTO group): wells were filled with complete culture medium containing sodium urate, and BaTiO3 nanoparticles at an optimal concentration of 0.2 mg / mL were added; Sodium urate + US group (US group): wells were treated with ultrasonic water bath (10~150 kHz, 5... The complete culture medium containing sodium urate was sonicated for 30 minutes in an ultrasonic water bath (0~1000W), and then the sonicated complete culture medium containing sodium urate was added to the wells; Sodium urate + BaTiO3 + US group (BTO + US group): The complete culture medium containing sodium urate and 0.2 mg / mL BaTiO3 nanoparticles was sonicated for 30 minutes in an ultrasonic water bath, and then the sonicated complete culture medium containing sodium urate and barium titanate nanoparticles was added to the wells. A total of five groups were incubated for 24 hours, and then various experimental data were explored and detected.

[0102] (2) Immunofluorescence staining experiment A. Seed Raw264.7 cells in confocal dishes, 2 × 10⁶ cells per well. 5 Cells were then treated with the Sham group (blank control group), sodium urate + PBS group, sodium urate + BaTiO3 group, sodium urate + US group, and sodium urate + BaTiO3 + US group and incubated for 24 h. The culture medium was then removed and the cells were washed three times with PBS. B. Fixation: Fix cells with 4% paraformaldehyde for 15 min, and wash with PBS 3 times after fixation; C. Permeability: After incubating the cells with PBS (containing 0.1% Triton X-100) for 10 minutes, wash them 3 times with PBS; D. Blocking: Incubate cells with 5% BSA at room temperature for 30 min, then wash 3 times with PBS; E. Primary antibody incubation: Dilute the antibody (1:200) with primary antibody dilution buffer, incubate the antibody at 4°C for 15 h, and wash the cells with PBS 5 times for 5 min each time; F. Secondary antibody incubation: Dilute goat anti-rabbit secondary antibody (or mouse antibody, 1:200) with diluent and incubate in the dark for 2-3 hours; G. Finally, add DAPI to each well, let it stand at room temperature for 15 minutes, and then take fluorescence images under dark conditions.

[0103] To investigate the in vitro anti-gout inflammation activity of BaTiO3 nanoparticles, we recreated the excessive activation of macrophages in a gout inflammation model by stimulating RAW264.7 cells with sodium urate. We then used immunofluorescence assays to detect changes in the fluorescence intensity of IL-1β, TNF-α, iNOS, and NLRP3.

[0104] from Figure 5 It can be seen that sodium urate-induced Raw264.7 cells (PBS) exhibited obvious inflammatory characteristics, with increased expression of inflammatory factors (IL-1β, TNF-α, iNOS, and NLRP3). However, neither ultrasound treatment without drug addition (sodium urate + US group) nor drug addition without ultrasound treatment (sodium urate + BaTiO3 group) had significant therapeutic effects on sodium urate-induced cells, and the fluorescence intensity of each inflammatory factor was basically no different from that of the sodium urate-treated model group.

[0105] The sodium urate + BaTiO3 + US treatment group significantly reduced the fluorescence intensity of IL-1β, TNF-α, iNOS and NLRP3, indicating that BaTiO3 nanoparticles can effectively inhibit the expression of inflammatory factors and alleviate gout inflammation under ultrasound.

[0106] (3) In vitro protein immunoblotting A. For the five groups of cells under different conditions mentioned above, discard the culture medium, gently wash the cells 2-3 times with PBS, add 750 μL of pre-cooled PBS solution to each well, collect the cells on ice using a cell scraper and transfer them to 1.5 mL centrifuge tubes, centrifuge at 1100 rpm and 4°C for 5 minutes; discard the supernatant, add 200 μL of prepared lysis buffer to each tube, shake vigorously for 30 seconds and place on ice, incubate for a total of 30 minutes, shaking every 10 minutes to ensure complete protein lysis; centrifuge at 14000 rpm for 15 minutes at 4°C, transfer the supernatant and store on ice, which is the sample containing protein.

[0107] B. Protein concentration detection and denaturation treatment The total protein concentration was determined using the BCA method, following the instructions of the Beyotime® BCA Protein Assay kit. Standards with gradient concentrations of 0 μg / μL, 0.025 μg / μL, 0.05 μg / μL, 1 μg / μL, 2 μg / μL, 3 μg / μL, 4 μg / μL, and 5 μg / μL were prepared in advance. Working solutions were prepared according to the kit instructions and added to each well of a 96-well plate (200 μL). Then, 20 μL of diluted protein sample or gradient concentration protein standards were added to the working solution, and the plates were incubated at 37°C for 25 minutes. The absorbance at 562 nm was measured using a microplate reader, and the protein concentration was plotted and calculated using a standard curve. To standardize protein concentrations across groups, an appropriate amount of protein sample was thoroughly mixed with RIPA lysis buffer and 5× loading buffer, boiled at 100°C for 5–10 minutes to denature the protein, and stored at -80°C, avoiding repeated freeze-thaw cycles.

[0108] C. Western blot analysis of the protein expression levels of inflammation-related markers IL-1β, TNF-α, and iNOS. (a) SDS-PAGE gel electrophoresis ① Gel Preparation: Based on the molecular weight of the target protein, prepare a lower separating gel of appropriate concentration according to the instructions. Mix all reagents according to the specified ratio and inject the gel into a double-layered glass plate until the gel surface reaches the upper part of the glass plate. Slowly add isopropanol, flatten and seal the gel surface. After the separating gel has fully solidified, discard the isopropanol. Next, prepare the upper stacking gel according to the instructions. Mix the gel solution and inject it into the glass plate above the separating gel until the edge of the glass plate. Scrape off any air bubbles at the edge while inserting the comb. Let the gel stand until it has fully solidified. Carefully check the quality of the gel. Once confirmed to be correct, place the glass plate into the electrophoresis tank and electrophoresis solution. Gently remove the comb and use the tip of the pipette to blow away any remaining stacking gel and air bubbles from the wells. ② Protein loading: Calculate the protein loading volume based on the measured protein concentration, and balance the protein loading buffer to ensure consistent loading volume in each lane. Use a micropipette to add the processed sample and pre-stained protein marker sequentially to the loading wells, generally ensuring a consistent total protein volume in each well; ③ Electrophoresis: Use a constant voltage of 60V for electrophoresis. When the protein markers are separated by bromophenol blue, adjust the voltage to a constant voltage of 120V. During electrophoresis, carefully observe the protein markers. Stop electrophoresis after the target proteins have separated, remove the glass plate, and cut off the stacking gel.

[0109] (b) Transfer membrane Select a PVDF membrane with an appropriate pore size based on the target protein molecular weight and cut it to the appropriate size. Soak it in methanol for 20 seconds. Place each component in the following order: negative electrode (black plate of the transfer clamp), sponge, filter paper, gel, PVDF membrane, filter paper, sponge, and positive electrode (white plate of the transfer clamp), taking care to avoid generating air bubbles. Fix the transfer clamp on the transfer device and immerse it in pre-cooled transfer buffer. Perform constant current transfer at 250mA at 4°C.

[0110] (c) Antibody incubation and protein development Remove the PVDF membrane from the conversion clamp. Cut the PVDF membrane containing the target protein, referencing the protein marker location, and label it. Wash with TBS buffer for 5 minutes, repeating 3 times. Block the membrane by shaking in 5% BSA solution at room temperature for 1 hour. Dilute the antibody 1:1000 with 5% BSA solution and incubate the PVDF membrane overnight at 4°C with the antibody dilution. The next day, wash the PVDF membrane with TBST solution (TBS solution containing 1‰ Tween-20) for 15 minutes, repeating 3 times. Place the washed membrane in an appropriate concentration of secondary antibody dilution solution and incubate with shaking at room temperature for 2 hours. After incubation, wash the membrane 3 times with TBST for 10-15 minutes each time to remove unbound secondary antibody. Using an ECL kit, evenly drop the mixture onto the membrane, place the membrane in a dark box for exposure, capture the chemiluminescent signal, and then develop.

[0111] from Figure 6 It can be seen that after sodium urate stimulation (MSU), the protein expression of IL-1β, TNF-α, and iNOS in cells was upregulated. When sodium urate solution was treated with ultrasound alone or with only the drug added without ultrasound treatment, and subsequently co-cultured with cells, the protein expression levels of IL-1β, TNF-α, and iNOS in the cells did not change significantly; however, the protein expression levels of IL-1β, TNF-α, and iNOS in the sodium urate + BaTiO3 + US treatment group decreased significantly, indicating that BaTiO3 nanoparticles can effectively alleviate gout inflammation under ultrasound.

[0112] 6. Treatment of mice with gout (1) Construction of an in vivo acute gout model A No. 4 needle was used to inject sodium urate crystal dispersion into the joint cavity along the medial side of the rat's Achilles tendon, following strict aseptic technique. The injection standard was the swelling of the contralateral joint capsule. Twelve hours later, swelling of the rat's right posterior ankle joint and restricted joint movement were observed, confirming the successful establishment of the model.

[0113] (2) Treatment A. Drug preparation: Dissolve 20mg BaTiO3 nanoparticles in 100mL of physiological saline, then disperse them thoroughly by sonication, and then draw up the drug preparation using a sterile 1mL insulin syringe.

[0114] B. Injection Procedure: The operator gently stabilizes the mouse's ankle joint with one hand, while holding the syringe in the other, inserting the needle into the joint space (usually in the depression on the lateral side of the ankle joint). Slowly advance the needle until a feeling of resistance is felt, aspirate to ensure no blood is drawn, and then slowly and evenly inject the medication into the joint cavity. After injection, quickly withdraw the needle and gently press the puncture site with a sterile cotton swab for a moment to prevent leakage.

[0115] C. Parameter Settings: Set the parameters of the ultrasound therapy device (frequency: 1MHz; duty cycle: 50%; intensity: 1.0W / cm). 2 ).

[0116] D. Ultrasound therapy: One hour after the intra-articular injection, the affected ankle joint area is disinfected. Sufficient ultrasound coupling agent is evenly applied to the surface of the ultrasound probe and the skin of the mouse's ankle joint. Then, the ultrasound probe is gently placed on the swollen area of ​​the mouse's ankle joint and slowly rotated in a spiral motion in the joint area to ensure that the ultrasound waves are evenly stimulated throughout the entire area. The ultrasound therapy can be stopped after 15 minutes.

[0117] E. End of treatment: After the ultrasound stimulation is completed, gently wipe away the residual coupling agent on the mouse skin with sterile gauze, and then observe the pain.

[0118] (3) Relief of apparent swelling and recovery of mobility after treatment: Gouty rats were randomly divided into four groups (blank control group, BaTiO3 group, ultrasound group, and BaTiO3 + ultrasound group), with 6 rats in each group. They were given intra-articular injection of the drug and ultrasound stimulation for 15 minutes 1 hour after drug administration. The circumference of the right posterior ankle joint at the same position was measured with calipers every 6 hours, and the mean value was taken for 3 consecutive times. Red and blue dyes were applied to the paws of the normal hind limbs and gouty hind limbs of the rats, respectively, and the rats were forced to walk on white strips of paper. The degree of matching between the red and blue footprints of the rats was observed and analyzed to determine their gait. The activity ability and activity duration of the rats were continuously recorded using an infrared camera to evaluate the overall recovery effect.

[0119] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0120] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of a piezoelectric material in the preparation of a drug for treating gout, characterized in that, The active pharmaceutical ingredient of the drug includes the piezoelectric material, which is barium titanate; The application includes: delivering the drug to the lesion site, applying ultrasound stimulation to the lesion site, and the drug stimulating the degradation of uric acid by active species in the body.

2. The application according to claim 1, characterized in that, The in vivo active species include at least one of hydroxyl radicals and superoxide radicals.

3. The application according to claim 1, characterized in that, In the drug, the mass concentration of the piezoelectric material is 0.01~10 mg / mL.

4. The application according to claim 1, characterized in that, The delivery method includes local injection.

5. The application according to claim 1, characterized in that, The conditions for the ultrasound stimulation include: frequency: 1 kHz to 10 MHz; duty cycle: 1% to 100%; intensity: 0.01 to 10 W / cm². 2 Time: 0.1~1000 minutes.

6. The method for preparing the piezoelectric material in any one of claims 1-5, characterized in that, The preparation method includes the following steps: Synthesis of sodium tritiate: Titanium dioxide was dispersed in sodium hydroxide solution, and the mixture was stirred and sonicated alternately to obtain a suspension. The suspension was subjected to hydrothermal reaction, and the precipitate was collected by centrifugation and washed to obtain sodium tritiate. Synthesis of layered titanate nanotubes: Sodium tritiate was dispersed in hydrochloric acid solution, stirred, centrifuged to collect the precipitate, washed, and dried to obtain layered titanate nanotubes; Synthesis of piezoelectric materials: Barium hydroxide octahydrate is dispersed in water with nitrogen continuously introduced, and the mixture is stirred and sonicated alternately. After the nitrogen is stopped, layered titanate nanotubes are added by stirring and sonicating alternately. The mixture undergoes a hydrothermal reaction, and the precipitate is collected by centrifugation, washed, and dried to obtain the piezoelectric material.

7. The preparation method according to claim 6, characterized in that, According to the dosage ratio, the titanium dioxide : the sodium hydroxide solution : the hydrochloric acid solution is (0.01~10) g : (0.01~100) mL : (0.01~350) mL; According to the mass ratio, the octahydrate barium hydroxide : the layered titanate nanotubes are (0.01~10):

1.

8. The preparation method according to claim 6, characterized in that, In the synthesis of sodium trititanate, the hydrothermal reaction is carried out at a temperature of 120~300℃ for a time of 1~120h. In the synthesis of the piezoelectric material, the hydrothermal reaction temperature is 120~300℃ and the time is 1~12h.

9. A drug for treating gout, characterized in that, It includes a pharmaceutically active ingredient and pharmaceutically acceptable excipients, wherein the pharmaceutically active ingredient includes a piezoelectric material as described in any one of claims 1-5 or a piezoelectric material obtained by the preparation method described in claims 6-8.

10. The medicament according to claim 9, characterized in that, The excipients include at least one of the following: excipients, lubricants, antioxidants, preservatives, binders, fillers, or thickeners.

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