PH response hydrogel microneedle and preparation method thereof

Abstract

The invention discloses a pH response hydrogel microneedle, which comprises a microneedle substrate, microneedle tips are arranged on the microneedle substrate, the height of the microneedle tips is 100-1600 microns, the diameter of the tip ends of the microneedle tips is 5-15 microns, and the density of the microneedle tips is 200-1000 pieces per square centimeter. The preparation method comprises the following steps: preparing a hyaluronic acid solution for the microneedle substrate, preparing a methacrylated chondroitin sulfate solution for the microneedle tip, preparing methacrylated orthoester, preparing orthoester, preparing a non-steroidal anti-inflammatory drug solution, preparing a microneedle tip solution, preparing the microneedle and demolding the microneedle to prepare the complete soluble microneedle patch. The invention has the advantages of high drug efficiency, high efficiency in dissolving fat / water drugs, and long-acting slow release.

Description

Technical Field

[0001] The present invention relates to the technical field of hydrogel microneedle preparation, in particular to a pH-responsive hydrogel microneedle and a preparation method thereof. Background Art

[0002] Rheumatoid arthritis (RA) is a disease characterized by erosive arthritis that primarily destroys joint cartilage and bone, ultimately leading to joint deformity and loss of function. The goals of RA treatment are to relieve symptoms, control disease progression, and improve patients' quality of life.

[0003] The main treatment strategies for rheumatoid arthritis include: (1) Drug treatment: Traditional synthetic DMARDs: such as methotrexate, which is the basic drug for the treatment of RA; biological agents: such as anti-tumor necrosis factor (TNF) inhibitors, interleukin-6 (IL-6) inhibitors, etc.; targeted synthetic DMARDs: such as JAK inhibitors.

[0004] (2) Non-drug treatment: including physical therapy, rehabilitation training and patient education.

[0005] (3) Surgical treatment: For patients who are ineffective with drug treatment and whose joint function is severely impaired, surgical intervention may be considered.

[0006] Traditional drug delivery methods primarily include oral administration, but these methods can pose drawbacks such as gastrointestinal reactions, low drug utilization, and significant toxic side effects. Patches and ointments can reduce drug delivery efficiency, while injections carry the risk of infection and can cause severe stinging. Microneedles, a combination of subcutaneous injection and transdermal patches, offer advantages such as simplicity, painlessness, safety, rapid onset of action, accurate drug delivery, avoidance of the first-pass effect, and high drug bioavailability. Currently, the main types of microneedles on the market include solid microneedles, hollow microneedles, coated microneedles, dissolving microneedles, and hydrogel microneedles. After penetration into the skin, hydrogel microneedles absorb tissue fluid and swell without dissolving, enabling controlled drug release. The degree of swelling of hydrogel microneedles is related to the degree of cross-linking in the material, which in turn affects the drug release rate. For example, patent application publication number CN115337530B discloses colchicine hydrogel microneedles and their preparation method. Acrylamide, N,N-bis(acryloyl)cysteamine, and Irgacure 2959 are used as matrix materials, and photocrosslinking is performed to produce hydrogel microneedles. A colchicine solution is added to the hydrogel, and the microneedles are allowed to swell and then air-dried. Patent application publication number CN118717641B discloses soluble gel microneedles, their preparation method, and applications. These microneedles, using sialic acid and gelatin as their primary matrix materials, exhibit excellent mechanical and biocompatibility, and slowly dissolve after insertion into the skin. However, all of these hydrogel microneedles share common challenges: they cannot stably and efficiently load lipid-soluble drugs, and they cannot achieve precise, long-term sustained release. Therefore, existing hydrogel microneedles require further improvement. Summary of the Invention

[0007] The purpose of the present invention is to make up for the above-mentioned deficiencies and disclose to the public a pH-responsive hydrogel microneedle and its preparation method with high drug delivery efficiency, high efficiency in dissolving fat / water drugs, and long-term sustained release.

[0008] The technical solution of the present invention is achieved as follows: A pH-responsive hydrogel microneedle comprises a microneedle substrate, on which a microneedle tip is arranged, wherein the microneedle tip has a height of 100 to 1600 microns, a tip diameter of 5 to 15 microns, and a density of 200 to 1000 microneedle tips per square centimeter.

[0009] Preferably, the microneedle tip is in the shape of a regular quadrangular pyramid or a cone.

[0010] A method for preparing pH-responsive hydrogel microneedles comprises the following steps: S1. Prepare hyaluronic acid solution for microneedle substrate: weigh hyaluronic acid powder into a sterile container and add ultrapure water to dissolve it into a hyaluronic acid solution; S2. Preparation of methacrylated chondroitin sulfate solution for microneedle tips: Weigh methacrylated chondroitin sulfate powder and photoinitiator Irgacure 2959 powder in a sterile container, add ultrapure water to dissolve and prepare methacrylated chondroitin sulfate solution and Irgacure 2959 solution; S3. Prepare a methacrylic acid acylated orthoester monomer; weigh diglycerol and trimethyl orthoformate, add a catalyst to react to obtain 2.3.2.1 4,4'-dimethyleneoxy-bis-(2-methoxy-1,3-dioxolane); add trifluoroacetamide and pyridinium p-toluenesulfonate to react to obtain 2.3.2.2 4,4'-dimethyleneoxy-bis-[2,2,2-trifluoro-N-(2-methoxy-1,3-dioxolane-4-ethylidene)acetamide]; then add tetrahydrofuran to react to obtain 2.3.2.3 4,4'-dimethyleneoxy-bis-(2-aminoethoxy-1,3-dioxolane); and finally react with methacrylic anhydride to obtain a methacrylic acid acylated orthoester monomer; S4. Preparation of orthoester: Weigh diglycerol and triethyl orthoacetate, add a catalyst and react to obtain orthoester; S5. Preparing a nonsteroidal anti-inflammatory drug solution: Weighing nonsteroidal anti-inflammatory drug powder into a centrifuge tube, adding the orthoester prepared in step S4, and sonicating to dissolve it to form an orthoester / anti-inflammatory drug mixed solution; S6. Preparing a microneedle tip solution: adjusting the pH of the methacrylated chondroitin sulfate solution prepared in step S2 to alkaline, and then thoroughly mixing it with the methacrylated orthoester prepared in step S3 and the solution prepared in step S5, and stirring evenly to obtain a microneedle tip solution; S7, preparing microneedles: adding the microneedle tip solution prepared in step S6 to the microneedle mold, preparing the tip by vacuum negative pressure and removing bubbles, then curing it into a gel by ultraviolet light to form a microneedle tip, then adding the microneedle base solution, and drying at room temperature to obtain the microneedle; S8. Microneedle demoulding: The microneedle patch prepared on the microneedle mold is demoulded using the air blowing demoulding method to obtain a complete soluble microneedle patch.

[0011] Preferably, the hyaluronic acid used in the preparation of the hyaluronic acid solution for the microneedle substrate in step S1 is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine, with a molecular formula (C 14 H 21 NO 11 ) n, molecular weight is 200-400 KDa.

[0012] Preferably, the chondroitin sulfate solution used in preparing the microneedle tip in step S2 is a glycosaminoglycan composed of N-acetyl-D-galactosamine and D-glucuronic acid repeating disaccharide units, and some sugar residues have sulfate groups, and the molecular formula is (C 14 H 21 NO 14 S)n, molecular weight is 25-40 KDa.

[0013] Preferably, in step S5, the concentration of the anti-inflammatory drug is 16 mg / ml-22 mg / ml.

[0014] Preferably, in step S6, the volume ratio of methacryloyl chondroitin sulfate: methacryloyl orthoester: orthoester is 5:1:0.5-5:1:0.75.

[0015] Preferably, in step S2, the concentration of methallylated chondroitin sulfate is 100 mg / ml-200 mg / ml.

[0016] Preferably, in step S2, the concentration of the photoinitiator Irgacure 2959 is 4 w / v%-10 w / v%.

[0017] Preferably, in step S6, the pH value of the methacrylated chondroitin sulfate solution is adjusted to 8.

[0018] The advantages of the present invention compared with the prior art are: (1) The organohydrogel microneedle patch of the present invention has a simple preparation method, good needle-forming properties, excellent mechanical and biocompatibility, and can be directly administered through the skin. The microneedle patch does not reach the dermis and is almost painless.

[0019] (2) The organohydrogel microneedle patch of the present invention comprises a microneedle tip body formed from methacryloyl chondroitin sulfate and methacryloyl orthoester, which are covalently cross-linked under the action of ultraviolet light and the photoinitiator Irgacure 2959 to form a hydrogel. Because the methacryloyl orthoester is pH-sensitive and easily degrades in acidic environments, it can effectively respond to the acidic environment of the inflammatory site and achieve a controlled release effect by varying the degree of cross-linking.

[0020] (3) The organohydrogel microneedle patch of the present invention can efficiently and stably load fat-soluble drugs by loading orthoesters.

[0021] (4) The organohydrogel microneedle patch of the present invention can be used to treat rheumatoid arthritis. The orthoester can efficiently load celecoxib and load it into the hydrogel microneedles formed by photocrosslinking of methacryloyl chondroitin sulfate and methacryloyl orthoester under the action of a photoinitiator. The microneedles act on the inflammation site, and the methacryloyl orthoester can respond to the acidic environment of the inflammation site. The hydrogel microneedles are gradually eroded, and the orthoester escapes from the hydrogel. In the acidic environment, accompanied by the degradation of the orthoester, the drug is released into the inflammation site to treat joint inflammation. The pH-responsive organohydrogel microneedles prepared by the present invention are suitable for treating rheumatoid arthritis and have good clinical application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is the hydrogen nuclear magnetic resonance spectrum of CSMA in the present invention (the deuterated reagent is D2O); Figure 2 is the hydrogen nuclear magnetic resonance spectrum of OE in the present invention (the deuterated reagent is DMSO); Figure 3 This is a schematic diagram of the preparation of the hydrogel in Example 1 of the present invention; Figure 4 The XRD spectra of the components of the hydrogel microneedle in Example 1 of the present invention are shown in FIG. Figure 5 FTIR images of the components of the hydrogel microneedles in Example 1 of the present invention; Figure 6 This is a screening diagram of the components of the hydrogel microneedles of the present invention; Figure 7 The actual image and AFM, SEM, and LSCM images of the hydrogel microneedle of Example 1 of the present invention are shown; Figure 8 This is a diagram showing the mechanical strength evaluation of the hydrogel microneedles in Example 1 of the present invention; Figure 9 This is a diagram showing the ability of the hydrogel microneedles in Example 1 of the present invention to penetrate the skin; Figure 10 This is a diagram showing the recovery of skin pores after the hydrogel microneedles of Example 1 of the present invention are inserted into the skin; Figure 11 Graph showing the in vivo solubility of different types of hydrogel microneedles in the present invention; Figure 12 It is the standard curve diagram of CXB in the present invention; Figure 13 Graph showing drug release of the hydrogel microneedles prepared in the present invention under different pH conditions; Figure 14 This is a diagram showing the evaluation results of cell activity of the hydrogel microneedles prepared in the present invention; Figure 15This is a cell live-dead staining image of the hydrogel microneedle prepared in the present invention; Figure 16 This is a graph showing the blood compatibility evaluation results of the hydrogel microneedles prepared in the present invention; Figure 17 This is a graph showing the results of in vitro anti-inflammatory performance evaluation of the hydrogel microneedles prepared in the present invention; Figure 18 Schematic diagram of the hydrogel microneedles prepared in the present invention for treating rheumatoid arthritis mice; Figure 19 This is a graph showing the changing trend of the paw width of mice in each group after the three hydrogel microneedles prepared by the present invention were used to treat inflammation mice; Figure 20 This is a graph showing the changing trend of clinical scores of the paws of mice in each group after the three hydrogel microneedles prepared by the present invention were used to treat inflammation mice; Figure 21 This is a graph showing the weight change trend of mice in each group after the three hydrogel microneedles prepared by the present invention were used to treat inflammation mice; Figure 22 This is a photo of the hydrogel microneedles prepared in the present invention applied to the soles of mice after the treatment of rheumatoid arthritis in mice; Figure 23 This is a Micro CT image of the paw of a mouse after the hydrogel microneedle prepared in the present invention was used to treat rheumatoid arthritis in the mouse; Figure 24 H&E staining and Safranin O Fast Green staining of mouse joints after treatment of rheumatoid arthritis in mice with the hydrogel microneedles prepared by the present invention; Figure 25 This is an immunohistochemical analysis of mouse joints after the hydrogel microneedles prepared in the present invention were used to treat rheumatoid arthritis in mice. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below with reference to the accompanying drawings: A pH-responsive hydrogel microneedle comprises a microneedle substrate, on which a microneedle tip is arranged, wherein the microneedle tip has a height of 100 to 1600 microns, a tip diameter of 5 to 15 microns, and a density of 200 to 1000 microneedle tips per square centimeter.

[0024] Preferably, the microneedle tip is in the shape of a regular quadrangular pyramid or a cone.

[0025] A method for preparing pH-responsive hydrogel microneedles comprises the following steps: S1. Preparation of hyaluronic acid solution for microneedle substrate: In a clean bench, weigh hyaluronic acid powder into a sterile container, add ultrapure water to dissolve and prepare a hyaluronic acid solution; the hyaluronic acid used in the hyaluronic acid solution is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine, with a molecular formula (C 14 H 21 NO 11 ) n, molecular weight is 200-400 KDa.

[0026] S2. Preparation of methacrylated chondroitin sulfate solution for microneedle tips: In a clean bench, weigh methacrylated chondroitin sulfate powder and photoinitiator Irgacure 2959 powder into a sterile container, add ultrapure water to dissolve and prepare methacrylated chondroitin sulfate solution and Irgacure 2959 solution; the chondroitin sulfate used in the chondroitin sulfate solution is a glycosaminoglycan composed of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid, with some sugar residues having sulfate groups, and the molecular formula is (C 14 H 21 NO 14 Preferably, the concentration of methacrylated chondroitin sulfate is 100 mg / ml-200 mg / ml, and the concentration of photoinitiator Irgacure 2959 is 4 w / v%-10 w / v%.

[0027] S3. Preparation of methacrylic orthoester monomer: Weigh diglycerol and trimethyl orthoformate, add a catalyst to react to obtain 2.3.2.1 4,4′-dimethyleneoxy-bis-(2-methoxy-1,3-dioxolane); add trifluoroacetamide and pyridinium p-toluenesulfonate to react to obtain 2.3.2.2 4,4′-dimethyleneoxy-bis-[2,2,2-trifluoro-N-(2-methoxy-1,3-dioxolane-4-ethylidene)acetamide]; then add tetrahydrofuran to react to obtain 2.3.2.3 4,4′-dimethyleneoxy-bis-(2-aminoethoxy-1,3-dioxolane); and finally react with methacrylic anhydride to obtain methacrylic orthoester monomer. S4. Preparation of orthoester: Weigh diglycerol and triethyl orthoacetate, add a catalyst and react to obtain orthoester; S5. Prepare a nonsteroidal anti-inflammatory drug solution: In a clean bench, weigh nonsteroidal anti-inflammatory drug powder into a centrifuge tube, add the orthoester prepared in step S4, and sonicate to dissolve it to form an orthoester / anti-inflammatory drug mixed solution; preferably, the concentration of the nonsteroidal anti-inflammatory drug is 16 mg / ml-22 mg / ml.

[0028] S6. Prepare a microneedle tip solution: In a clean bench, adjust the pH value of the methacrylated chondroitin sulfate solution prepared in step S2 to alkaline, and then fully mix it with the methacrylated orthoester in step S3 and the solution prepared in step S5, and stir evenly to obtain a microneedle tip solution; preferably, the volume ratio of methacrylated chondroitin sulfate: methacrylated orthoester: orthoester is 5:1:0.5-5:1:0.75; the pH value of the methacrylated chondroitin sulfate solution is adjusted to 8.

[0029] S7. Prepare microneedles: Add the microneedle tip solution prepared in step S6 into the microneedle mold, prepare the tip by vacuum negative pressure and remove bubbles, then cure it into glue by ultraviolet light to form a microneedle tip, then add the microneedle base solution, and dry it at room temperature to obtain the microneedle.

[0030] S8. Microneedle demoulding: The microneedle patch prepared on the microneedle mold is demoulded using the air blowing demoulding method to obtain a complete soluble microneedle patch.

[0031] The present invention is further described below through specific examples and comparative examples.

[0032] The present invention provides a method for preparing a pH-responsive hydrogel microneedle, comprising the following steps: The present invention first prepares methacrylated chondroitin sulfate (CSMA), methacrylated orthoester (OEMA) and orthoester (OE), specifically as follows: 1. Preparation of methacrylated chondroitin sulfate (CSMA): Weigh 1 g of chondroitin sulfate and dissolve it in 50 ml of ultrapure water. After complete dissolution, add 0.114 mol of methacrylic anhydride (MAA) dropwise to the CS solution, shielding the solution from light. Then, add 5 mol / L NaOH dropwise to adjust the pH to 8.0. The mixture is stirred at room temperature for two hours, then stirred in an ice bath in the dark for 24 hours. The mixture is precipitated with a large amount of ethanol and ultrasonically washed several times with a large amount of ethanol. The resulting product is dried in a vacuum oven at room temperature to obtain the product CSMA, whose structural formula is: Its H NMR spectrum (deuterated reagent is D2O) is shown in Figure 1 , confirmed by NMR DS=47%.

[0033] 2. Preparation of methacrylamide orthoester monomer (OEMA): Under nitrogen, add 27.02 g (0.16 mol) of diglycerol, 150 mL of acetonitrile, 138.04 g (1.3 mol) of trimethyl orthoformate, and 0.56 g of p-toluenesulfonic acid monohydrate (p-TSA·H2O) to a 250 mL three-necked round-bottom flask. Stir and react overnight at room temperature. Add a few drops of triethylamine to the round-bottom reaction flask to terminate the transesterification, and completely remove the acetonitrile by rotary evaporation. Dissolve the crude product in 250 mL of ethyl acetate and extract twice with 400 mL of 10% Na2CO3. Collect the organic phase, dry over anhydrous Mg2SO4, and filter and concentrate to obtain 2.3.2.1 4,4 ， -dimethyleneoxy-bis-(2-methoxy-1, 3-dioxolane) (denoted as product 1); 12.48 g (0.05 mol) of product 1, 17.24 g (0.11 mol) of trifluoroacetamide, and 251 mg (1 mol) of pyridinium p-toluenesulfonate were weighed and added to a 100 mL eggplant-shaped reaction flask. The mixture was stirred vigorously in an oil bath at 130°C for 8 h. After the reaction, the mixture was cooled to room temperature and dissolved in 200 mL of ethyl acetate. The mixture was extracted twice with 6% NaHCO₃ solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to yield 2.3.2.2 4, 4 ， -dimethyleneoxy-bis-[2, 2, 2-trifluoro-N-(2-methoxy-1, 3-dioxolane-4-ethylidene)acetamide] (denoted as product 2); Place 20.06 g (0.04 mol) of product 2 in a 250 mL single-necked round-bottom flask. Dissolve product 2 completely in 100 mL of tetrahydrofuran (THF) containing triethylamine. Add 135 mL of 3 M NaOH solution and stir for 24 h. Completely remove the THF by rotary evaporation. Extract three times with approximately 150 mL of CH2Cl2. The collected dichloromethane (DCM) phases were dried over anhydrous magnesium sulfate, filtered, and concentrated to yield 2.3.2.3 4, 4. ， -dimethyleneoxy-bis-(2-aminoethoxy-1,3-dioxolane) (referred to as product 3); Weigh 2.17 g (7.04 mmol) of product III into a 250 mL three-necked round-bottom flask, then add 4.25 g (42.00 mmol) of triethylamine and dissolve thoroughly in 80 mL of anhydrous CH2Cl2. Under nitrogen, slowly add 3.24 g (21.02 mmol) of methacrylic anhydride dropwise in an ice bath. Let the reaction react for 12 h. Dichloromethane was removed by vacuum distillation, dissolved in 100 mL of ethyl acetate, and extracted twice with 10% Na2CO3 solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was then separated and purified using a silica gel column to obtain the final product, methacryloyl orthoester monomer (OEMA), with the structural formula: .

[0034] 3. Preparation of Orthoester (OE): Diglycerol and trimethyl orthoacetate were weighed in a 1:3 molar ratio in a 250 ml three-necked flask. An appropriate amount of catalyst, p-toluenesulfonic acid monohydrate (p-TSA·H2O), was added. 50 ml of anhydrous dichloromethane was added and stirred overnight at room temperature under a nitrogen atmosphere. After the reaction, a few drops of triethylamine were added. The mixture was poured into a 250 ml round-bottom flask and rotary evaporated at 40°C for about half an hour to remove the trimethyl orthoacetate and dichloromethane. After rotary evaporation, the mixture was dissolved in approximately 120 ml of ethyl acetate and poured into a separatory funnel. The mixture was slowly quenched three times with saturated sodium carbonate. Finally, the mixture was dried over 20 g of anhydrous magnesium sulfate for at least 8 hours, shaking the mixture periodically. The anhydrous magnesium sulfate was removed by filtration using a sand core funnel. The ethyl acetate was then removed by rotary evaporation at 50°C for 1 hour to obtain the product, OE, with the following structural formula: Its H NMR spectrum (D2O) is shown in Figure 2 .

[0035] Example 1 (1) Microneedle substrate raw materials: hyaluronic acid (HA). The hyaluronic acid used in the hyaluronic acid solution for microneedle substrate is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine. The molecular formula is (C 14 H 21 NO 11 ) n, with a molecular weight of 200-400 KDa. The prepared hyaluronic acid solution has excellent viscoelasticity and biocompatibility for the microneedle substrate.

[0036] (2) Raw materials for microneedle tips: methacryloyl chondroitin sulfate (CSMA), methacryloyl orthoester (OEMA), Irgacure 2959. The structure of chondroitin sulfate used in the preparation of microneedle tips is a glycosaminoglycan composed of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid. Some sugar residues may have sulfate groups. The molecular formula is (C 14 H 21 NO 14S)n, molecular weight is 25-40 KDa.

[0037] (3) Nonsteroidal anti-inflammatory drugs: Celecoxib (CXB), molecular formula C 17 H 14 Celecoxib (F3N3O2S, molecular weight 387.31) selectively inhibits cyclooxygenase-2 (COX-2), reducing prostaglandin synthesis and thereby alleviating inflammation. Its analgesic effects are suitable for treating a variety of pain conditions, including acute and chronic pain. In some cases, celecoxib can also reduce fever.

[0038] A method for preparing pH-responsive hydrogel microneedles, the preparation process is as follows Figure 3 As shown, the following steps are included: S1. Preparation of HA solution for microneedle substrate: In a clean bench, weigh HA into a sterile container, add ultrapure water and dissolve it to obtain a HA solution with a concentration of 60 mg / ml.

[0039] S2. Preparation of CSMA solution for microneedle tips: In a clean bench, CSMA and Irgacure 2959 were weighed separately in sterile containers and dissolved in ultrapure water to prepare a mixed solution of 200 mg / ml CSMA solution and 1 w / v% Irgacure 2959.

[0040] S3. Prepare OEMA solution: Under nitrogen, add 27.02 g (0.16 mol) of diglycerol, 150 mL of acetonitrile, 138.04 g (1.3 mol) of trimethyl orthoformate, and 0.56 g of p-toluenesulfonic acid monohydrate (p-TSA·H2O) to a 250 mL three-necked round-bottom flask in sequence. Stir and react overnight at room temperature. Add a few drops of triethylamine to the round-bottom reaction flask to terminate the transesterification. Rotary evaporation is then used to completely remove the acetonitrile. Dissolve the crude product in 250 mL of ethyl acetate and extract twice with 400 mL of 10% Na2CO3. Collect the organic phase, dry it over anhydrous Mg2SO4, and filter and concentrate to obtain 2.3.2.1 4, 4 ， -dimethyleneoxy-bis-(2-methoxy-1, 3-dioxolane) (denoted as product 1); 12.48 g (0.05 mol) of product 1, 17.24 g (0.11 mol) of trifluoroacetamide, and 251 mg (1 mol) of pyridinium p-toluenesulfonate were weighed and added to a 100 mL eggplant-shaped reaction flask. The mixture was stirred vigorously in an oil bath at 130°C for 8 h. After the reaction, the mixture was cooled to room temperature and dissolved in 200 mL of ethyl acetate. The mixture was extracted twice with 6% NaHCO₃ solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated to yield 2.3.2.2 4, 4 ，-dimethyleneoxy-bis-[2, 2, 2-trifluoro-N-(2-methoxy-1, 3-dioxolane-4-ethylidene)acetamide] (denoted as product 2); Place 20.06 g (0.04 mol) of product 2 in a 250 mL single-necked round-bottom flask. Dissolve product 2 completely in 100 mL of tetrahydrofuran (THF) containing triethylamine. Add 135 mL of 3 M NaOH solution and stir for 24 h. Completely remove the THF by rotary evaporation. Extract three times with approximately 150 mL of CH2Cl2. The collected dichloromethane (DCM) phases were dried over anhydrous magnesium sulfate, filtered, and concentrated to yield 2.3.2.3 4, 4. ， -dimethyleneoxy-bis-(2-aminoethoxy-1,3-dioxolane) (referred to as product 3); 2.17 g (7.04 mmol) of product III was weighed and placed in a 250 mL three-necked round-bottom flask. 4.25 g (42.00 mmol) of triethylamine was added and fully dissolved in 80 mL of anhydrous CH2Cl2. Under nitrogen, 3.24 g (21.02 mmol) of methacrylic anhydride was slowly added dropwise in an ice bath. The reaction was allowed to react for 12 hours. Dichloromethane was removed by vacuum distillation, and the product was dissolved in 100 mL of ethyl acetate and extracted twice with 10% Na2CO3 solution. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and concentrated. The crude product was then separated and purified on a silica gel column to obtain the final product, methacryloyl orthoester monomer (OEMA).

[0041] S4. Preparation of OE: 20.00 g (0.12 mol) of diglycerol was reacted with 58.58 g (0.36 mol) of triethyl orthoacetate and 0.1145 g (0.6 mmol) of p-toluenesulfonic acid monohydrate (p-TSA·H2O) to obtain the orthoester (OE).

[0042] S5. Prepare OE / CXB solution: In a clean bench, weigh CXB into a sterile container, add the OE prepared in step S4 and sonicate to dissolve it, to prepare an OE / CXB solution with a concentration of 22 mg / ml.

[0043] S6. Prepare a microneedle tip solution: In a clean bench, adjust the CSMA solution prepared in step S2 to pH 8, and then fully mix it with the OEMA in step S3 and the OE / CXB solution prepared in step S5 in a volume ratio of 5:1:0.5, and stir evenly to obtain a microneedle tip solution.

[0044] S7. Preparation of microneedles: In an ultra-clean workbench, pour the mixed emulsion prepared in step S6 into the microneedle mold. After the liquid surface is filled evenly and leveled, place the mold in a vacuum drying oven and evacuate the mold to allow the raw material solution to enter the needle tip. Then remove the bubbles on the liquid surface and the needle tip solution remaining on the mold surface. Irradiate with ultraviolet light for 5 minutes and photocuring into glue to form the needle tip. Spread the HA solution prepared in step S1 on the substrate and dry it at room temperature for 12 hours to obtain the microneedle.

[0045] S8 Microneedle demolding: The prepared hydrogel microneedle patch was demolded from the microneedle mold using the air blowing demolding method to obtain a complete soluble microneedle patch (denoted as CAOO-CXB MNs).

[0046] The structures of CS, CSMA, OEMA, OE, CSMA / OEMA, and CSMA / OEMA / OE were characterized by X-ray diffraction effect method. The obtained XRD patterns are shown in the figure below. Figure 4 XRD analysis of the sample structure shows a broad diffraction peak at 20.97° for CS and a broad peak at 21.75° for CSMA. The characteristic peaks shifted, confirming the successful synthesis of CSMA. Cross-linking of CSMA with OEMA resulted in a sharper peak and shifted to 22.01°, confirming successful cross-linking.

[0047] The structures of CS, CSMA, OEMA, OE, CSMA / OEMA, and CSMA / OEMA / OE were characterized by FT-IR method. The obtained Fourier transform infrared spectra are shown in the figure below. Figure 4 As shown in Figure 5, the characteristic peaks of CSMA Vc=o (1727 cm-1), Vc=c (1640 cm-1) and the characteristic peak of OEMA Vc=c (1640 cm-1) appear, which proves that CSMA and OEMA were successfully prepared.

[0048] Screening of raw materials for hydrogel microneedle components Figure 6 , the photoinitiator content is as follows Figure 6 In Figure a, the CSMA solid content is 20 mg / ml, and the concentrations of the photoinitiator are screened as 1.2 w / v%, 1 w / v%, 0.8 w / v%, 0.6 w / v%, and 0.4 w / v%. After light irradiation and gelation, the gelation state is observed and the elastic modulus of the gel is detected by rheometer. As the photoinitiator content increases, the elastic modulus of the hydrogel increases. However, when the photoinitiator concentration is 1.2 w / v%, the prepared microneedles will have photoinitiator precipitation, exceeding the maximum solubility of the hydrogel; the content of different proportions of OEMA is screened as follows Figure 6In Figure b, the CSMA solid content is 200 mg / ml, the concentration of the photoinitiator is 1 w / v%, and the volume ratios of CSMA to OEMA are 1:1, 5:1, 10:1, 20:1, and 30:1, respectively. The OEMA content is screened by gelation state and rheometer test of the elastic modulus of the gel. As the OEMA content increases, the elastic modulus of the hydrogel increases. When the volume ratio exceeds 5:1, OEMA self-crosslinks and cannot form a gel. To further determine the content of OE loaded in the gel, see Figure 6 In Figure c, with a fixed photoinitiator content of 1 w / v%, VCSMA:VOEMA:VOE ratios of 5:1:1, 5:1:0.75, and 5:1:0.5 were screened. The gelation state of each component was observed, and the gelled hydrogels were screened using rheometer testing. The hydrogel elastic modulus was highest at a volume ratio of 5:1:0.5, making the hydrogel microneedles more likely to meet the mechanical strength required for skin penetration. Following these screening steps, a VCSMA:VOEMA:VOE ratio of 5:1:0.5 was determined for the MNs, with a photoinitiator content of 1 w / v%.

[0049] Microneedle morphology and structure Figure 7 The morphology of the microneedles was observed using a camera, an atomic force microscope, and a scanning electron microscope. Rhodamine B was dissolved into the microneedles and the morphology of the microneedles was observed using a laser confocal microscope. Through the above methods, it was observed that the prepared organic hydrogel microneedles had a neat microneedle array, complete needle tips, and a density of 400 / cm 2 The shape of the microneedle tip is a regular quadrangular pyramid, which proves that the microneedle preparation is successful.

[0050] Microneedle mechanical strength test Figure 8 As shown, the prepared hydrogel microneedles were cut into a 5*5 array, and the mechanical strength of the array was tested with a universal testing machine to calculate the mechanical strength of each needle tip of the hydrogel microneedle. The test results show that the mechanical strength of a single needle tip of the hydrogel microneedle CAO MNs is greater than the minimum force of 0.1 N required to penetrate the skin. The mechanical strength of CAOO MNs in Example 1 was compared. The test results show that due to the cross-linking of OEMA in the hydrogel, the elastic modulus of the hydrogel increases, and the mechanical strength of the hydrogel microneedles will be further increased to meet the requirements of insertion into the skin.

[0051] Test of the ability of microneedles to penetrate the skin and skin recovery: Mice were anesthetized with isoflurane, and then the backs of the mice were depilated. The prepared hydrogel microneedles were added to the skin of the mice's backs and wrapped with sterile tape to prevent them from falling off. The skin was then pressed for ten minutes to allow the microneedles to fully penetrate the mouse skin. Figure 10 As shown in the figure, after the microneedles were peeled off, a clear array of needle tips was left on the back of the mouse. Figure 11As shown, the back skin of mice gradually recovered over time and was completely recovered at 4 h, with no obvious damage on the skin surface.

[0052] Example 2 A pH-responsive hydrogel microneedle, comprising the following raw materials: (1) Microneedle base material: hyaluronic acid (HA); (2) Microneedle tip materials: methacryloyl chondroitin sulfate (CSMA), methacryloyl orthoester (OEMA), Irgacure 2959, orthoester (OE); (3) Nonsteroidal anti-inflammatory drugs: celecoxib (CXB).

[0053] S1. Preparation of HA solution for microneedle substrate: In a clean bench, weigh HA powder into a sterile container, add ultrapure water and dissolve to obtain a HA solution with a concentration of 60 mg / ml.

[0054] S2. Preparation of CSMA solution for microneedle tips: In a clean bench, CSMA and Ireg2959 were weighed separately into sterile containers, and ultrapure water was added to dissolve them to prepare a mixed solution with a concentration of 20 mg / ml CSMA and 1 w / v% Irgacure2959, respectively.

[0055] S3. Prepare OEMA solution.

[0056] S4. Preparation of OE: 20.00 g (0.12 mol) of diglycerol was reacted with 58.58 g (0.36 mol) of triethyl orthoacetate and 0.1145 g (0.6 mmol) of p-toluenesulfonic acid monohydrate (p-TSA·H2O) to obtain the orthoester (OE).

[0057] S5. Prepare OE / CXB solution: In a clean bench, weigh CXB into a sterile container, add the orthoester OE prepared in step S4 and sonicate to dissolve it, to prepare an OE / CXB solution with a concentration of 22 mg / ml.

[0058] S6. Preparing a microneedle tip solution: uniformly mixing the mixed solution prepared in step S2, the OEMA solution prepared in step S3, and the OE / CXB solution prepared in step S5 in a volume ratio of 10:1:0.5 to form an emulsion to obtain a microneedle tip solution.

[0059] S7. Prepare microneedles: In an ultra-clean workbench, pour the mixed emulsion prepared in step S5 into the microneedle mold. After the liquid surface is filled evenly and leveled, place the mold in a vacuum drying oven and evacuate the vacuum to allow the raw material solution to enter the needle tip. Then remove the bubbles on the liquid surface and the needle tip solution remaining on the mold surface. Irradiate with ultraviolet light for 5 minutes and photocuring into glue to form the needle tip. Spread the microneedle base solution prepared in step S1 on the base and dry at room temperature for 12 hours to obtain the microneedle.

[0060] S8. Microneedle demoulding: Use the air blowing demoulding method to demould the prepared hydrogel microneedle patch from the microneedle mold to obtain a complete soluble microneedle patch.

[0061] Example 3 A pH-responsive hydrogel microneedle, comprising the following raw materials: (1) Microneedle base material: hyaluronic acid (HA); (2) Microneedle tip materials: methacryloyl chondroitin sulfate (CSMA), methacryloyl orthoester (OEMA), Irgacure 2959, orthoester (OE) (3) Nonsteroidal anti-inflammatory drugs: celecoxib (CXB) S1. Preparation of HA solution for microneedle substrate: In a clean bench, weigh HA into a sterile container and add ultrapure water to dissolve it to obtain a HA solution with a concentration of 60 mg / ml.

[0062] S2. Preparation of CSMA solution for microneedle tips: In a clean bench, CSMA and Irgacure 2959 were weighed separately into sterile containers, and ultrapure water was added to dissolve them to prepare a mixed solution of 20 mg / ml CSMA and 0.8 w / v% Irgacure 2959.

[0063] S3. Prepare OEAM solution.

[0064] S4. Preparation of OE: 20.00 g (0.12 mol) of diglycerol was reacted with 58.58 g (0.36 mol) of triethyl orthoacetate and 0.1145 g (0.6 mmol) of p-toluenesulfonic acid monohydrate (p-TSA·H2O) to obtain the orthoester (OE).

[0065] S5. Prepare OE / CXB solution: In a clean bench, weigh CXB into a sterile container, add the OE prepared in step S4 and dissolve it to prepare an OE / CXB solution with a concentration of 22 mg / ml.

[0066] S6. Preparing a microneedle tip solution: uniformly mixing the mixed solution prepared in step S2, the OEMA prepared in step S3, and the OE / CXB solution prepared in step 3 in a volume ratio of 5:1:0.5 to form an emulsion to obtain a microneedle tip solution.

[0067] S7. Prepare microneedles: In an ultra-clean workbench, pour the mixed emulsion prepared in step S6 into the microneedle mold. After the liquid surface is filled evenly and leveled, place the mold in a vacuum drying oven and evacuate the mold to allow the raw material solution to enter the needle tip. Then remove the bubbles on the liquid surface and the needle tip solution remaining on the mold surface. Irradiate with ultraviolet light for 5 minutes and photocuring into glue to form the needle tip. Spread the microneedle base solution prepared in step S1 on the base and dry it at room temperature for 12 hours to obtain the microneedle.

[0068] S8. Microneedle demoulding: The prepared hydrogel microneedle patch is demoulded from the microneedle mold using an air blowing demoulding method to obtain a complete hydrogel microneedle patch.

[0069] Comparative Example 1 An organic hydrogel microneedle patch, its raw material components: (1) Microneedle base material: hyaluronic acid (HA).

[0070] (2) Microneedle tip material: chondroitin sulfate (CS).

[0071] S1. Preparation of HA solution for microneedle substrate: In a clean bench, weigh HA into a sterile container and add ultrapure water to dissolve it to obtain a HA solution with a concentration of 60 mg / ml.

[0072] S2. Preparation of CS solution for microneedle tips: In a clean bench, weigh CS powder into a sterile container and add ultrapure water to dissolve it to prepare a CS solution with a concentration of 20 mg / ml.

[0073] S3. Preparation of microneedles: In a clean bench, pour the CS solution prepared in step S2 into the microneedle mold. After the liquid surface is evenly filled and leveled, place the mold in a vacuum drying oven and evacuate the mold to allow the raw material solution to enter the needle tip. Then remove the bubbles on the liquid surface, and then spread the HA solution prepared in step S1 on the substrate. Dry at room temperature for 12 h to obtain microneedles.

[0074] S4. Microneedle demoulding: The prepared hydrogel microneedle patch was demoulded from the microneedle mold using the air blowing demoulding method to obtain a complete soluble microneedle patch (denoted as C MNs).

[0075] Comparative Example 2 An organic hydrogel microneedle patch, its raw material components: (1) Microneedle base material: hyaluronic acid (HA); (2) Microneedle tip materials: methacryloyl-chondroitin sulfate (CSMA), Irgacure 2959; S1. Preparation of HA solution for microneedle substrate: In a clean bench, weigh HA into a sterile container and add ultrapure water to dissolve it to obtain a HA solution with a concentration of 60 mg / ml.

[0076] S2. Preparation of CSMA solution for microneedle tips: In a clean bench, CSMA and Irgacure 2959 were weighed separately in sterile containers and dissolved in ultrapure water to prepare a mixed solution with a concentration of 20 mg / ml CSMA and 1 w / v% Irgacure 2959, respectively.

[0077] S3. Preparation of microneedles: In an ultra-clean workbench, pour the mixed solution prepared in step S2 into the microneedle mold. After the liquid surface is filled evenly and leveled, place the mold in a vacuum drying oven and evacuate the mold to allow the raw material solution to enter the needle tip. Then, remove bubbles on the surface of the liquid, irradiate with ultraviolet light for 5 minutes, and photocure into glue to form the needle tip; spread the HA solution prepared in step S1 on the substrate and dry it at room temperature for 12 hours to obtain the microneedle.

[0078] S4. Microneedle demoulding: The prepared hydrogel microneedle patch was demoulded from the microneedle mold using the air blowing demoulding method to obtain a complete soluble microneedle patch (denoted as CA MNs).

[0079] Comparative Example 3 (1) Microneedle base material: hyaluronic acid (HA); (2) Microneedle tip materials: methacryloyl chondroitin sulfate (CSMA), Irgacure 2959, orthoester (OE) (3) Nonsteroidal anti-inflammatory drugs: celecoxib (CXB) S1. Preparation of HA solution for microneedle substrate: In a clean bench, weigh HA into a sterile container and add ultrapure water to dissolve it to obtain a HA solution with a concentration of 60 mg / ml.

[0080] S2. Preparation of CSMA solution for microneedle tips: In a clean bench, CSMA and Irgacure 2959 were weighed separately in sterile containers, and ultrapure water was added to dissolve them to prepare a mixed solution of 20 mg / ml CSMA and 10 mg / ml Ireg2959.

[0081] S3. Preparation of OE: 20.00 g (0.12 mol) of diglycerol was reacted with 58.58 g (0.36 mol) of triethyl orthoacetate and 0.1145 g (0.6 mmol) of p-toluenesulfonic acid monohydrate (p-TSA·H2O) to obtain the orthoester (OE).

[0082] S4. Prepare OE / CXB solution: In a clean bench, weigh CXB into a sterile container, add the OE prepared in step S3 and sonicate to dissolve it, to prepare an OE / CXB solution with a concentration of 22 mg / ml.

[0083] S5. Prepare a microneedle tip solution: In a clean bench, fully mix the mixed solution prepared in step S2 and the OE / CXB mixed solution prepared in S4 at a volume ratio of 5:0.5 to form an emulsion.

[0084] S6. Prepare microneedles: In an ultra-clean workbench, pour the mixed solution prepared in S5 into the microneedle mold. After the liquid surface is evenly filled and leveled, place the mold in a vacuum drying oven to evacuate the solution to allow the solution to enter the needle tip. Then remove the bubbles on the surface of the liquid. Irradiate with ultraviolet light for 5 minutes to photocurate into glue to form the needle tip. Spread the HA solution prepared in S1 on the substrate and dry it at room temperature for 12 hours to obtain the microneedle.

[0085] S7. Microneedle demolding: The prepared hydrogel microneedle patch was demolded from the microneedle mold using the air blowing demolding method to obtain a complete soluble microneedle patch (denoted as CAO-CXB MNs).

[0086] Characterization of the physicochemical properties of hydrogel microneedles: In vivo degradation of hydrogel microneedles: The AIA modeling method was used to establish a mouse rheumatoid arthritis model. Then different types of microneedle patches were attached to the inflamed areas of the mouse joints. The microneedle patches were removed after 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 36h, and 48h, and the dissolution of the microneedles was observed under a microscope.

[0087] Establish a CXB standard curve: dissolve 50 mg of CXB in 1 mL of PBS buffer solution to prepare a 50 mg / mL CXB solution, and use 0.2 w / v% SDS to assist solubilization. Then, dilute the solution by half with PBS buffer solution to prepare CXB solutions with different concentrations. Use a microplate reader to measure the absorbance at a wavelength of 270 nm.

[0088] Drug release from hydrogel microneedles: Using the hydrogel microneedles prepared in the present invention, two different hydrochloric acid buffers (pH 7.4 and pH 5.8) were set up, with 0.2 w / v% SDS as a cosolvent. A phosphate buffer at pH 5.8 was used to simulate the inflammatory microenvironment. The buffer was placed in a diffusion cell and slowly stirred at 37°C to simulate the in vivo environment. A sealing film was used to simulate the skin. The hydrogel microneedles prepared in the present invention were then inserted into the sealing film and secured on the back with transparent tape. The needle tips were then immersed in the buffer. At different time points of 0.5 h, 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h, 1 ml of buffer was aspirated at different time points, and then 1 ml of buffer was added. The drug concentration in the buffer was then measured by HPLC to calculate the drug release content of the hydrogel microneedles in the present invention.

[0089] Biosafety evaluation of hydrogel microneedles: Cytocompatibility testing: The safety of the hydrogel microneedles was assessed using MTT assays and live-dead double staining. Mouse fibroblasts (L929) were seeded into 96-well and 24-well culture plates containing MEM medium and incubated in a sterile incubator for 12 hours to allow complete cell attachment. The old culture medium was then removed and the plates were washed twice with PBS buffer. Fresh culture medium containing the hydrogel microneedle extract was then added. Cells cultured in MEM medium in the 96-well plates served as a positive control, while cells cultured in pure culture medium served as a negative control. The plates were incubated in a sterile incubator for 24 hours. 20 μl of MTT solution (5 mg / ml) was added to each well of the 96-well plates and incubated for an additional 4 hours. The MTT solution was removed, and 200 μl of DMSO was added to each well. After 15 minutes, the absorbance at 570 nm was measured and cell viability was calculated. Add 500 μl of buffer containing 1% calcein AM and 0.1% iodide (PI) to each well of a 24-well plate. Incubate at room temperature for 30 min. Wash with PBS to remove background color, and observe cell viability and cell death using an inverted fluorescence microscope. Perform three replicates per group.

[0090] Hemocompatibility testing: 500 μl of mouse whole blood was collected in an anticoagulant tube and then dispersed into 5 ml of PBS buffer. The blood was centrifuged at 2500 rpm for 5 minutes, the supernatant discarded, and the cells were washed three times with PBS. The cells were then resuspended in 10 ml of saline to prepare a red blood cell suspension for later use. Next, 500 μl of red blood cell suspension was mixed with different types of hydrogel microneedle extracts. A negative control group consisted of 500 μl of red blood cell suspension plus 500 μl of saline, while a positive control group consisted of 500 μl of red blood cell suspension plus ultrapure water. The cells were incubated at 37°C and 120 rpm for 1 hour, and the supernatant was centrifuged and the absorbance at 540 nm was measured to calculate the hemolysis rate. Three parallel experiments were performed for each group.

[0091] To evaluate the in vitro anti-inflammatory properties of the hydrogel microneedles, RAW cells were seeded in a 6-well culture plate containing DMEM and cultured in a sterile incubator for 24 hours. The supernatant was discarded and the cells were washed twice with PBS. The culture medium was replaced with 450 ml of fresh culture medium containing 1 mg / ml LPS for 24 hours. Simultaneously, 50 μl of different hydrogel microneedle extracts were added for intervention. A 50 μl PBS control group was used for incubation in a sterile incubator for 24 hours. The culture medium was collected and the levels of three inflammatory factors were analyzed using ELISA kits.

[0092] Evaluation of hydrogel microneedle therapy for rheumatoid arthritis: Using ICR mice, after one week of acclimation, 50 μl of complete Freund's adjuvant was injected orally into the paw. Seven days later, another 50 μl complete Freund's adjuvant was injected for a booster immunization. Two weeks later, significant paw swelling developed, confirming successful modeling. Following modeling, different microneedles were applied to the lesion site on the paw every two days. The model group served as a control group. Weight, paw width, and clinical arthritis scores were recorded every three days. Following treatment, the paws of the mice underwent microCT scanning, Safranin O Fast Green staining, HE staining, and immunohistochemical analysis to evaluate the in vivo anti-inflammatory effects of the hydrogel microneedles.

[0093] Figure 11 The in vivo degradation diagram of the hydrogel microneedle prepared in the present invention is shown by Figure 11 It can be seen from the in vivo degradation of the hydrogel microneedles that the microneedles of comparative example 1 will degrade in 2 hours under an inflammatory environment, and comparative example 2 will degrade in 48 hours because the cross-linking degree of the hydrogel increases and the modulus increases after photocrosslinking. Example 1 will degrade in 36 hours because the elastic modulus of the gel will be reduced after loading with OE. When OEMA is added, OEMA will cross-link with CSMA. Because OEMA is pH sensitive and can respond to the environment of the inflammatory site, the hydrogel swells and degrades. As OE escapes, the drug is further released, and Example 2 is completely degraded in 4 hours.

[0094] Figure 12 This is the standard curve of CXB. Figure 13 The hydrogel microneedle drug release results prepared in the present invention are as follows: Figure 13 It can be seen that the drug release of Example 1 and Comparative Example 3 under different pH conditions is faster in Example 1 than in Comparative Example 3. Example 1 completely releases the drug within 24 hours in an environment of pH 5.8. Due to the presence of OEMA, the hydrogel network structure can respond to the acidic environment of the inflammatory site, and is destroyed faster, thereby promoting faster drug release.

[0095] Figure 14 、 Figure 15 and Figure 16 The hydrogel prepared in the present invention is used to prove the biosafety assessment results. Figure 14 The results of the cytotoxicity experiments of hydrogel microneedles showed that the cell survival rates of different hydrogel microneedles were all greater than 80%. Figure 15 The fluorescence photographs also showed that the cells basically exhibited green fluorescence (green: living cells; red blood cells: dead cells), indicating that the hydrogel microneedles prepared in Example 1 and Comparative Examples 2 and 3 all had good cell compatibility. Figure 16 The results of the hydrogel microneedle hemolysis experiment showed that in the positive control group treated with deionized water, red blood cells ruptured, a large amount of hemoglobin was released, and the supernatant was red. However, there was no significant difference between the hydrogel microneedles prepared in Example 1 and Comparative Examples 2 and 3 and the negative control group after treatment, and the supernatant was clear. The OD value of each experimental group was detected using a multifunctional microplate reader at a wavelength of 540 nm. The hemolysis rate of each group was less than 5%, indicating that no obvious hemolysis occurred and had good blood compatibility.

[0096] Figure 17 The results of the in vivo anti-inflammatory effect of the hydrogel microneedles prepared in the present invention are as follows. LPS induces M0 macrophages to differentiate into M1 macrophages, and the expression of pro-inflammatory factors such as TNF-α, IL-1β, and IL-6 is upregulated. After co-incubation of macrophages with different microneedle extracts, the levels of the three inflammatory factors in Example 1 were significantly reduced, far lower than those in Comparative Examples 2 and 3. This proves that the OEMA-crosslinked hydrogel microneedles in Example 1 can better promote drug release, thereby having a good anti-inflammatory effect in vitro.

[0097] Figure 18 This is a schematic diagram of mouse modeling and treatment of rheumatoid arthritis using the hydrogel microneedles prepared in the present invention.

[0098] Figure 19 、 Figure 20 and Figure 21 These are the experimental results of treating rheumatoid arthritis with the hydrogel microneedles prepared in the present invention. Figure 19 and Figure 20As shown by the changing trends of the mouse paw width and clinical scores during microneedle treatment, the hydrogel microneedles in Example 1 and Comparative Examples 2 and 3 all have certain anti-inflammatory and detumescent abilities. The hydrogel microneedles in Example 1 can respond to the inflammatory microenvironment of the arthritis site, better promote the release of drugs, and have the most significant effect in relieving joint swelling. Figure 21 Figure 3 shows the changes in body weight of mice in each group during the treatment period. The body weight of mice in each hydrogel microneedle treatment group remained basically stable, indicating that the mice were in good health.

[0099] Figure 22 and Figure 23 This is a real picture of the mouse's paw and a Micro CT scan image. Figure 22 The actual picture of the sole of the foot shows that the hydrogel microneedles in Example 1 and Comparative Examples 2 and 3 all have a certain effect in treating rheumatoid arthritis, and the pH-responsive hydrogel microneedles in Example 1 have the strongest anti-inflammatory and detumescent ability and the best effect in treating rheumatoid arthritis. Figure 23 Micro CT scanning was used to observe the bone damage and healing of the soles of the mice. The results showed that the joint structure of the soles of the mice in the normal group was intact and the joint surface was smooth, while the joints of the mice in the model group were eroded and the joint surface was rough, proving that the RA model was successfully established. After different types of microneedle treatments, the joints of the mice recovered to varying degrees. The joint surface of the control group 2 was still rough with no obvious signs of recovery. In control group 3, the joint bone damage was slightly repaired and the roughness of the joint surface was improved. The joint repair of the mice treated with the hydrogel microneedles in Example 1 was the best. Figure 24 H&E and Safranin-O Fast Green staining were used to observe histopathological changes in the articular cartilage of the mouse knee joints. H&E staining revealed that, compared to the normal group, the articular cartilage surface in the model group was severely damaged, rough, and lesioned. The articular cartilage surfaces in Comparative Examples 2 and 3 were rough and slightly worn. However, compared to the model group, the articular cartilage surface roughness in Comparative Examples 2 and 3 improved, while the articular surface in Example 1 was smoother. Safranin-O Fast Green staining was used to assess changes in the extracellular cartilage matrix of the articular cartilage. Compared to the normal group, the model group showed less red staining, indicating less proteoglycan distribution and severe articular cartilage wear. After treatment with different microneedle treatments, related symptoms improved. The cartilage surface in Comparative Example 2 was slightly uneven, while the proteoglycan distribution in Comparative Example 3 was relatively uniform, resulting in a smoother cartilage surface. The articular cartilage surface in Example 1 was the smoothest, approaching that of the normal group. This demonstrates that the hydrogel microneedles prepared in Example 1 have a significant therapeutic effect on cartilage damage.

[0100] Figure 25The immunohistochemical analysis in the experiment was to detect three inflammatory factors, IL-1β, IL-6, and TNF-a, in the joints of mice. The effect of the microneedles in Example 1 on inhibiting the inflammatory factors was more obvious than that in Comparative Examples 2 and 3, and was closer to that in the normal group, proving that the pH-responsive hydrogel microneedles prepared in Example 1 had a good anti-inflammatory effect. The hydrogel microneedles formed by cross-linking methacrylated chondroitin sulfate with methacrylated orthoesters can meet the requirements of mechanical strength, respond to the joint microenvironment, and release the drug in a long-term manner. In addition, the bicyclic orthoester can dissolve the non-steroidal anti-inflammatory drug, celecoxib, and promote drug penetration, thereby better acting on the inflammatory site, reducing the inflammatory response, and promoting cartilage regeneration.

[0101] The pH-responsive hydrogel microneedles prepared by the present invention not only have good biosafety compared to traditional hydrogel microneedles, but can also respond to the microenvironment of the inflammatory site, while loading non-steroidal anti-inflammatory drugs, exerting anti-inflammatory effects, and promoting drug penetration, thereby achieving a sustained-release effect, which can better act on the inflammatory site, inhibit the secretion of inflammatory factors, reduce inflammatory reactions, and promote cartilage and bone repair.

[0102] The above are only preferred embodiments of the present invention and do not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A pH-responsive hydrogel microneedle, characterized by: The invention comprises a microneedle substrate, on which a microneedle tip is arranged. The height of the microneedle tip is 100-1600 microns, the tip diameter of the microneedle tip is 5-15 microns, and the density of the microneedle tip is 200-1000 microns per square centimeter.

2. The pH-responsive hydrogel microneedle according to claim 1, wherein: The microneedle tip is in the shape of a regular quadrangular pyramid or a cone.

3. A method for preparing pH-responsive hydrogel microneedles, characterized by: The following steps are involved: S1. Prepare hyaluronic acid solution for microneedle substrate: weigh hyaluronic acid powder into a sterile container and add ultrapure water to dissolve it into a hyaluronic acid solution; S2. Preparation of methacrylated chondroitin sulfate solution for microneedle tips: Weigh methacrylated chondroitin sulfate powder and photoinitiator Irgacure 2959 powder in a sterile container, add ultrapure water to dissolve and prepare methacrylated chondroitin sulfate solution and Irgacure 2959 solution; S3. Preparation of methacrylic orthoester monomer: Weigh diglycerol and trimethyl orthoformate, add a catalyst to react to obtain 2.3.2.1 4,4′-dimethyleneoxy-bis-(2-methoxy-1,3-dioxolane); add trifluoroacetamide and pyridinium p-toluenesulfonate to react to obtain 2.3.2.2 4,4′-dimethyleneoxy-bis-[2,2,2-trifluoro-N-(2-methoxy-1,3-dioxolane-4-ethylidene)acetamide]; then add tetrahydrofuran to react to obtain 2.3.2.3 4,4′-dimethyleneoxy-bis-(2-aminoethoxy-1,3-dioxolane); and finally react with methacrylic anhydride to obtain methacrylic orthoester monomer. S4. Preparation of orthoester: Weigh diglycerol and triethyl orthoacetate, add a catalyst and react to obtain orthoester; S5. Preparing a nonsteroidal anti-inflammatory drug solution: Weighing nonsteroidal anti-inflammatory drug powder into a centrifuge tube, adding the orthoester prepared in step S4, and sonicating to dissolve it to form an orthoester / anti-inflammatory drug mixed solution; S6. Preparing a microneedle tip solution: adjusting the pH of the methacrylated chondroitin sulfate solution prepared in step S2 to alkaline, and then thoroughly mixing it with the methacrylated orthoester prepared in step S3 and the solution prepared in step S5, and stirring evenly to obtain a microneedle tip solution; S7, preparing microneedles: adding the microneedle tip solution prepared in step S6 to the microneedle mold, preparing the tip by vacuum negative pressure and removing bubbles, then curing it into a gel by ultraviolet light to form a microneedle tip, then adding the microneedle base solution, and drying at room temperature to obtain the microneedle; S8. Microneedle demoulding: The microneedle patch prepared on the microneedle mold is demoulded using the air blowing demoulding method to obtain a complete soluble microneedle patch.

4. The method for preparing a pH-responsive hydrogel microneedle according to claim 3, wherein: The hyaluronic acid used in the preparation of the hyaluronic acid solution for the microneedle substrate in step S1 is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine, with a molecular formula (C 14 H 21 NO 11 ) n, molecular weight is 200-400 KDa.

5. The method for preparing a pH-responsive hydrogel microneedle according to claim 3, wherein: The structure of the chondroitin sulfate used in the chondroitin sulfate solution for preparing the microneedle tip in step S2 is a glycosaminoglycan composed of repeating disaccharide units of N-acetyl-D-galactosamine and D-glucuronic acid, and some sugar residues have sulfate groups, and the molecular formula is (C 14 H 21 NO 14 S)n, molecular weight is 25-40 KDa.

6. The method for preparing a pH-responsive hydrogel microneedle according to claim 3, wherein: In step S5, the concentration of the nonsteroidal anti-inflammatory drug is 16 mg / ml-22 mg / ml.

7. The method for preparing a pH-responsive hydrogel microneedle according to claim 3, wherein: In step S6, the volume ratio of methacryloyl chondroitin sulfate: methacryloyl orthoester: orthoester is 5:1:0.5-5:1:0.

75.

8. The method for preparing a pH-responsive hydrogel microneedle according to claim 3, wherein: In step S2, the concentration of methallylated chondroitin sulfate is 100 mg / ml-200 mg / ml.

9. The method for preparing a pH-responsive hydrogel microneedle according to claim 3, wherein: In step S2, the concentration of the photoinitiator Irgacure 2959 is 4 w / v%-10 w / v%.

10. The method for preparing a pH-responsive hydrogel microneedle according to claim 3, wherein: In step S6, the pH value of the methacrylated chondroitin sulfate solution is adjusted to 8.

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