A dual-network hydrogel microneedle and a preparation method and application thereof

Abstract

The application discloses a kind of double-network hydrogel microneedles and preparation method and application thereof, and relates to the field of microneedle technology.The method comprises the following steps: dissolving methotrexate (MTX) in NaHCO3 solution, then adding anhydrous ethanol to mix, to obtain solution A;PVP K30 is dissolved in purified water to obtain solution B;MTX-NS is obtained by injecting solution A into solution B;PVP K30 is weighed and mixed with MTX-NS, and hyaluronic acid methacrylate (HAMA), methacrylated gelatin (GelMA) and photoinitiator (I2959) are added to prepare a pre-gel solution;after pouring the pre-gel solution on the microneedle mold, centrifugal treatment is carried out, then ultraviolet irradiation is carried out, the same pre-gel solution is poured again, and the MTX-NS double-network hydrogel microneedle is prepared by centrifugal treatment.

Description

Technical Field

[0001] This invention relates to the field of microneedle technology, specifically to a dual-network hydrogel microneedle, its preparation method, and its application. Background Technology

[0002] Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic, symmetrical, progressive polyarthritis. Its pathological features include abnormal proliferation of the synovial membrane, inflammatory cell infiltration, and the gradual irreversible erosion and destruction of cartilage and bone tissue. RA is characterized by high incidence and disability rates, imposing a heavy burden on patients and society. Currently, first-line drugs for the clinical treatment of RA, such as methotrexate (MTX), are mainly administered orally or via intra-articular injection. Oral administration suffers from problems such as the first-pass effect in the liver, large fluctuations in bioavailability, and significant systemic toxic side effects (such as liver damage and bone marrow suppression); while intra-articular injection is an invasive procedure requiring high levels of professional skill, resulting in poor patient compliance, and repeated punctures increase the risk of infection and joint damage. Traditional transdermal drug delivery (such as ointments) can avoid the first-pass effect and increase patient autonomy, but due to the highly efficient barrier of the stratum corneum, the transdermal efficiency of drugs, especially hydrophilic macromolecular drugs, is extremely low, making it difficult to achieve the required blood drug concentration or local drug exposure for treatment.

[0003] To overcome the barriers to transdermal drug delivery, microneedles (MNs) technology has emerged. Microneedle arrays can temporarily open microchannels in the stratum corneum in a minimally invasive manner, significantly improving drug transdermal efficiency. Among various types of microneedles, hydrogel microneedles exhibit outstanding advantages due to their unique "insertion-swelling" mechanism: after penetrating the skin, the needle absorbs interstitial fluid and swells, forming a continuous drug diffusion channel in the dermis, achieving sustained-release drug delivery over a longer period, making it particularly suitable for chronic diseases requiring long-term management, such as rheumatoid arthritis (RA). However, existing hydrogel microneedle technology still faces key technical challenges that urgently need to be addressed. 1. The contradiction between mechanical strength and skin puncture efficiency: To achieve effective puncture, microneedles need sufficient mechanical strength to resist the shear and compressive forces during insertion. Traditional single-network hydrogels (such as single hyaluronic acid or gelatin-based hydrogels) often have insufficient mechanical properties, making them prone to bending or breakage during puncture, leading to drug delivery failure. 2. Poor controllability of drug release behavior: The cross-linking density and pore structure of the hydrogel network directly affect drug release kinetics. Single-network structures are difficult to precisely control, often resulting in significant initial burst release of drugs, making it impossible to maintain stable, long-lasting therapeutic concentrations, and limiting the loading and protection capabilities for drugs (especially hydrophilic drugs or nanoparticle drug delivery systems). 3. Limited biological functionality: Many hydrogel materials only serve as physical carriers, lacking active biological regulatory functions. For the treatment of RA, ideal carrier materials should not only deliver drugs but also possess certain anti-inflammatory, lubricating, or tissue repair promotion potential.

[0004] To address these challenges, researchers have begun exploring dual-network hydrogel systems. These systems typically consist of an interpenetrating rigid, brittle first network and a flexible, tough second network, synergistically enhancing the material's mechanical properties (such as strength, toughness, and fatigue resistance). In the field of biomaterials, methacryloyl gelatin, due to its excellent photocrosslinking properties, biocompatibility, and extracellular matrix-like function, is often used to construct the first network, providing fundamental mechanical support. Methacrylyl hyaluronic acid, due to its inherent lubricating, anti-inflammatory, and cell behavior-regulating bioactivities, is often introduced as the second network to enhance the hydrogel's toughness and endow it with positive biological functions. However, the innovative application of the GelMA-HAMA dual-network hydrogel system to microneedle carriers for transdermal drug delivery in RA, and the systematic resolution of complex technical challenges such as its moldability, precise drug delivery control, and adaptability to the RA pathological microenvironment, lacks mature and optimized solutions in existing technologies. Especially... How to balance the dual-network ratio to achieve excellent microneedle puncture performance while ensuring the safety of hydrophobic drugs (such as MTX) or their derivatives? Highly efficient loading and programmed sustained release of nanocrystalline formulations The synergistic therapeutic effect of HA at the site of joint inflammation remains a significant technological gap in this field.

[0005] Therefore, there is an urgent need to develop a novel microneedle delivery system based on GelMA-HAMA dual-network hydrogel to overcome the shortcomings of existing single-network hydrogel microneedles and provide a new targeted therapy strategy for RA that combines high penetration, long-lasting sustained release, low side effects, and high patient compliance. Summary of the Invention

[0006] The present invention aims to provide a method for preparing a dual-network hydrogel microneedle, the method comprising the following steps: Step 1, dissolving methotrexate (MTX) in NaHCO3 solution, then adding anhydrous ethanol and mixing well to obtain solution A; Step 2, dissolving PVP K30 in purified water to obtain solution B; Step 3, injecting solution A into solution B under continuous stirring at 35°C, and continuing stirring to obtain MTX-NS for later use; Step 4, weighing PVP... K30 and MTX-NS are mixed and swelled in a 4°C refrigerator. Hyaluronic acid methacrylate (HAMA), methacrylamide gelatin (GelMA), and photoinitiator I2959 are added and stirred continuously in a 35°C water bath until the solution is uniformly mixed. The solution is then allowed to swell at room temperature to obtain a pregel solution. Step 5: After casting the pregel solution onto a microneedle mold, the solution is centrifuged and then irradiated with ultraviolet light. The same pregel solution is then poured in again and centrifuged to fully fill the mold backing area. Finally, the solution is irradiated with ultraviolet light to obtain MTX-NS loaded dual-network hydrogel microneedles.

[0007] Furthermore, in step three, when solution A is injected into solution B for mixing, the volume ratio between the two is 1:7, the volume ratio of MTX to PVP K30 is 2:3, and the stirring time is 62 min.

[0008] Furthermore, in step three, a centrifuge is used for stirring at a speed of 3120 r·min. -1， Centrifugation time was 10 minutes.

[0009] Furthermore, in step four, the mass ratio of methacrylamide hyaluronic acid (mHA) to methacrylamide gelatin (GelMA) is 1:2.

[0010] Furthermore, in step four, the mass ratio of methacrylamide hyaluronic acid (mHA) to polyvinylpyrrolidone K30 (PVPK30) is 1:5.

[0011] Furthermore, in step four, the mass ratio of methacrylamide hyaluronic acid to water is 1:36.

[0012] Furthermore, the centrifugation speed in both steps of step five was 3120 r·min. -1 The first UV crosslinking time was 86 min, and the second UV crosslinking time was 187 min.

[0013] Another objective of this invention is to provide a dual-network hydrogel microneedle prepared by the above-described preparation method.

[0014] The third objective of this invention is to provide the application of the above-mentioned dual-network hydrogel microneedles in products for the in vivo anti-inflammatory effect of ADA.

[0015] The present invention has the following technical effects: The present invention uses esterification and amidation reactions to synthesize hyaluronic acid methacrylate and methacrylated gelatin, and prepares blank double-network hydrogel microneedles by centrifugation molding method; MTX-NS is prepared by solvent-nonsolvent precipitation method, and then MTX-NS loaded double-network hydrogel microneedles are prepared by centrifugation molding method. Attached Figure Description

[0016] Figure 1 Flowchart for the fabrication of MTX-NS dual-network hydrogel microneedles; Figure 2 Download the appearance image of MTX-NS dual-network hydrogel microneedles for optical microscopy (a: before swelling, b: after swelling); Figure 3 Appearance of MTX-NS dual-network hydrogel microneedles downloaded for FESEM (a: ×20, b: ×90). Figure 4 To investigate the drug release rate in different microneedles using Rhodamine B as a simulated drug; Figure 5 The moisture absorption capacity of blank dual-network hydrogel microneedles and MTX-NS loaded dual-network hydrogel microneedles under different humidity conditions was measured (n=3) (a: relative humidity 33%, b: relative humidity 65%, c: relative humidity 96%). Figure 6 Image of skin puncture in rats with MTX-NS dual-network hydrogel microneedles (×10). Figure 7 Image showing skin healing in rats with MTX-NS dual-network hydrogel microparticles (×10, a: 0 min, b: 6 min, c: 12 min, d: 18 min, e: 24 min, f: 30 min). Figure 8 Download FESEM images of the appearance of MTX-NS dual-network hydrogel microneedles after skin swelling (a: 800 mm, b: 826±4.5 mm). Figure 9 Results of histological sections of rat skin tissue punctured by MTX-NS dual-network hydrogel microneedles; Figure 10 Cumulative release curves of MTX and MTX-NS dual-network hydrogel microneedles (±s, n=3). Figure 11 Cumulative transdermal permeability curves (±s, n=3) for MTX, MTX-NS, and MTX-NS-loaded dual-network hydrogel microneedles; Figure 12Results of hemolysis rates for different dual-network hydrogel microneedle solutions (±s, n=3) (a: B-HMN group, b: B-HMN group irradiated for 1 h, c: HMN group, d: HMN group irradiated for 1 h). Figure 13 The images show the morphological characteristics of RAW 264.7 cells before and after polarization (a: normal cells, b: cells after polarization). Figure 14 To polarize NO concentration in RAW 264.7 cells with different concentrations of LPS (±s, n=3) (24 h and 12 h, 24 h vs. 48 h), * P <0.05,** P <0.01, *** P <0.001); Figure 15 Cytotoxicity results for normal RAW264.7 and polarized RAW264.7 cells (±s, n=3) (a: B-HMN group, b: MTX group, c: HMN group, d: combined group, *P<0.05, ***P<0.001). Figure 16 Staining images of polarized RAW264.7 live cells by different concentrations of samples; Figure 17 shows the quantitative results of polarized RAW264.7 cell survival in each group (±s, n=3). Figure 18 The effect of each group on the levels of inflammatory factors induced by LPS in RAW 264.7 cells (±s, n=3) (a: IL-10, b: IL-1b, c: TNF-a, d: NO, ***) P <0.001); Figure 19 Western blots of iNOS and COX-2 proteins in RAW 264.7 cells for each group; Figure 20 The quantitative results of iNOS and COX-2 protein expression in RAW 264.7 cells for each group (±s, n=3) (compared with the model group, ** P <0.01); Figure 21 A graph showing the changes in arthritis scores in each group of rats (±s, n=3) (compared to the model group, *** P <0.001); Figure 22 Figure 5 shows the paw swelling of rats in each group on day 49 of day 32 (×5, a: blank group, b: model group, c: positive group, d: HMN group, e: ADA group, f: combined group). Figure 23 Figure 1 shows the changes in paw swelling in rats of each group (±s, n=3) (compared to the model group, ***P <0.001); Figure 24 Paw thickness changes in rats in each group (±s, n=3) (compared with the model group, *** P <0.001); Figure 25 X-ray images of the ankle joints of rats in each group (×10, a: blank group, b: model group, c: positive group, dHMN group, e: ADA group, f: combined group). Figure 26 H&E stained sections of rat ankle joints after treatment (a: blank group, b: model group, c: positive group, d: HMN group, e: ADA group, f: combined group). Figure 27 Images of Safranin-Fix Green stained sections of rat ankle joints after treatment (a: blank group, b: model group, c: positive group, d: HMN group, e: ADA group, f: combined group). Figure 28 Immunohistochemical analysis of inflammatory factors in the synovial tissue of the joint cavity; Figure 29 This is a semi-quantitative analysis diagram of inflammatory factors measured by immunohistochemistry. Detailed Implementation

[0017] Instruments: PDMS mold (needle height: 800 mm) - Micropoint Technologies PTE LTD, Singapore; TDZ5-WS benchtop low-speed centrifuge - Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; FTIR-650 Fourier transform infrared spectrometer - Tianjin Gangdong Technology Co., Ltd., China; DSC 200 F3 high-temperature differential thermal analyzer - Netzsch GmbH, Germany; FA2004N electronic analytical balance - Shanghai Shunyu Hengping Scientific Instrument Co., Ltd., China; DF-101S thermal collector-type constant temperature magnetic stirrer - Gongyi Yuhua Instrument Co., Ltd.; YB-P6 intelligent transdermal testing instrument - manufactured by Tianjin Pharmacopoeia Standard Instrument Factory; SHA-BA water bath constant temperature shaker - Changzhou Zhongbei Instrument Co., Ltd.; JSM-7800F field emission scanning electron microscope - JEOL Ltd., Japan.

[0018] Materials: Hyaluronic acid (HA), N,N-dimethylformamide (DMF), methacrylic anhydride (MA), gelatin, methotrexate (MTX), povidone K30 (PVP K30), and adalimumab were purchased from Shanghai Maclean Biotechnology Co., Ltd. (China). Sodium hydroxide (NaOH) was purchased from Merck Group (Germany). Photoinitiator I2959 was purchased from BASF AG (Germany). Anhydrous ethanol (analytical grade), sodium bicarbonate (NaHCO3), and phosphate buffered saline (PBS) (dry powder) were purchased from Hangzhou Shapu Biotechnology Co., Ltd. (China).

[0019] Example 1: Preparation and characterization of MTX-NS dual-network hydrogel microneedles 1. Preparation of MTX-NS dual-network hydrogel microneedles MTX active pharmaceutical ingredient was dissolved in NaHCO3 solution, and then anhydrous ethanol was added and mixed well to obtain solution A. PVP K30 was dissolved in purified water to obtain solution B. Under continuous stirring at 35°C, solution A was poured into solution B, and stirring was continued to obtain MTX-NS for later use.

[0020] Weigh an appropriate amount of PVP K30 and MTX-NS (the specific amount added should refer to the maximum blood concentration value of oral MTX in clinical practice), mix them, and place them in a 4°C refrigerator to swell. Add HAMA, GelMA, and photoinitiator I2959, and place them in a 35°C water bath with continuous stirring until the solution is uniformly mixed. Allow them to swell at room temperature, pour the above solution onto a microneedle mold, and centrifuge at 3120 r·min. -1 Centrifuge for 10 min to allow the solution to fill the bottom of the PDMS mold cavity and fully penetrate the mold needle tip. Then irradiate with ultraviolet light for a period of time, and then pour in the same solution and centrifuge at 3120 r·min. -1 Centrifuge for 10 min to fully fill the mold backing area, then irradiate with ultraviolet light to obtain MTX-NS dual-network hydrogel microneedles. A schematic diagram of the preparation is shown below. Figure 1 .

[0021] Observe the morphology of drug-loaded microneedles under an eyepiece, such as Figure 2 As shown, the microneedles had no air bubbles on their surface before swelling and had a square pyramidal structure; after swelling, the microneedles showed good swelling phenomenon, indicating that loading MTX-NS does not affect the swelling performance of the hydrogel.

[0022] The conductive adhesive was used to fix the MTX-NS dual-network hydrogel microneedles in the sample stage. The microneedle surface was then sputter-coated with gold. The microneedle sample stage was placed inside the FESEM, a vacuum was drawn, and the sample stage was rotated to a 30° angle with the horizontal plane. The field of view and magnification were adjusted to capture an electron microscope image of the microneedle surface. Figure 3As shown in the figure, under FESEM, the morphology of the MTX-NS dual-network hydrogel microneedles is as follows: they are pyramidal in shape, with sharp tips, complete array, no bending or breakage, and the morphology is the same as that of the blank microneedles.

[0023] Release characteristics of dual-network hydrogel microneedles: Agarose hydrogel was used to simulate skin to study the drug release characteristics of HAMA hydrogel microneedles, GelMA hydrogel microneedles, and dual-network hydrogel microneedles (prepared by mixing the two). Rhodamine B was used as a drug simulant. Agarose powder was dissolved in boiled purified water, allowed to cool, and agarose hydrogel was prepared. Rhodamine B-loaded hydrogel microneedles were pressed onto the surface of the agarose hydrogel, and the results were observed continuously for 7 days. The color depth of the agarose gel was observed by cutting it open daily.

[0024] like Figure 4 As shown, under conditions of 25℃ and 37℃, the three network structures of the microneedles had a consistent effect on the release of rhodamine B. With increasing release time, the diffusion rate and extent of rhodamine B in the dual-network hydrogel microneedles were significantly faster than those in the GelMA and HAMA hydrogel microneedles, and the released color deepened, indicating an increase in the amount of rhodamine B released. Furthermore, at the same release time, higher temperatures favored drug release. During the release of rhodamine B, the sufficient swelling phenomenon of the dual-network hydrogel microneedles further promoted the release rate and extent of rhodamine B; therefore, the dual-network hydrogel structure can alter the drug release rate.

[0025] Stability Study of Drug-Loaded Microneedles: Since the water content in HFMN affects the mechanical properties of microneedles, the stability of the hygroscopic capacity of MTX-NS dual-network hydrogel microneedles during storage and transportation was investigated. Blank hydrogel microneedles and MTX-NS dual-network hydrogel microneedles were stored for 14 days at relative humidityes of 33%, 65%, and 96%, respectively (magnesium chloride simulated a relative humidity of 33%, sodium nitrite simulated a relative humidity of 65%, and potassium sulfate simulated a relative humidity of 96%). The hydrogels were weighed every 48 hours. The hygroscopic capacity was calculated using Equation 1-1. ; Among them W 初始 and W 最终 These are the initial and final weights of the hydrogel, respectively.

[0026] Figure 5As shown, under three different humidity conditions, both the blank microneedles and the MTX-NS dual-network hydrogel microneedles exhibited good hygroscopic capacity of <15%. The hygroscopic capacity of the drug-loaded microneedles was similar to that of the blank microneedles, suggesting it would not affect the swelling rate determination. The swelling rate determination was performed according to the reference. The blank hydrogel microneedles and the MTX-NS dual-network hydrogel microneedles were weighed in a dry state (M0), then immersed in PBS solution at room temperature for 1, 2, 4, 8, 12, 18, 24, 32, 40, 50, 60, 90, 120, 150, and 180 min. After removal, the excess surface water was gently blotted with filter paper, and the microneedles were weighed again at each time point (M0). t ), calculate the expansion percentage, i.e. the equilibrium expansion percentage, see 1-2. ; Drug-loaded microneedle skin puncture study: Rat skin was harvested, subcutaneous fat was removed, and the stratum corneum of the rat skin was placed upwards. MTX-NS dual-network hydrogel microneedles were inserted perpendicularly to the rat skin, and force was applied to puncture the microneedles. After 1-2 minutes, the microneedle patch was removed, and the number of puncture sites on the rat skin was observed. The same MTX-NS dual-network hydrogel microneedle insertion treatment was performed on rats, and the microneedles were removed after 5 minutes. Skin healing was observed, and the skin puncture sites were photographed every 6 minutes until complete healing. Figure 6 and Figure 7 As shown.

[0027] Swelling of drug-loaded microneedles in rat skin Microneedles loaded with MTX-NS dual-network hydrogel were inserted into the skin of rat backs, fixed with medical tape for 10 minutes, and then removed. The swelling of the needles was observed under FESEM. Figure 8 As shown.

[0028] like Figure 9 As shown, the MTX-NS dual-network hydrogel microneedles left needle-like cavity channels in the rat skin, and the cavity had a rounded appearance. The channel was about 780 mm long, which is similar to the results of the experimentally prepared 800 mm high drug-loaded microneedles. This indicates that the MTX-NS dual-network hydrogel microneedles can pierce the stratum corneum of rat skin, swell in the skin, and form drug delivery channels.

[0029] In vitro release assay of drug-loaded microneedles The in vitro release rates of MTX and MTX-NS-loaded dual-network hydrogel microneedles were investigated using dialysis. 15 mg of MTX standard was accurately weighed and diluted to 22 mL with pH 7.4 PBS solution containing 1% SDS. This solution was transferred to a dialysis bag with a molecular weight cutoff of 14000 Da and placed in 300 mL of pH 7.4 PBS solution containing 1% SDS for constant temperature shaking. Several MTX-NS-loaded dual-network hydrogel microneedle patches were prepared to contain 15 mg of MTX, and the two layers of parafilm were punctured. ® After membrane preparation, the sample was floated in 300 mL of pH 7.4 PBS solution containing 1% SDS, with the needle tip in contact with the liquid surface, and subjected to isothermal shaking at a rate of 110 r / min. -1 The oscillation temperature was 37±0.5℃. Samples of 5.0 mL were taken at set times of 0.08 (5 min), 0.25 (15 min), 0.5 (30 min), 1, 2, 4, 6, 8, 12, 24, 48, and 72 h, with the same volume of release medium added simultaneously. The samples were analyzed using HPLC, and the cumulative drug release rate (%) was calculated to investigate its relationship with time (t, h). The cumulative release amount (Qn, mg·mL) was also determined. -1 Formula (1-3) for calculating cumulative release (%); Formula (1-4) for calculating cumulative release (%). ; ; Among them: Q n C represents the cumulative release at point i; n The concentration of TBH released during each sampling; C i Vi represents the concentration of the i-th sample; V0 represents the volume of the release medium; Vi represents the concentration of the i-th sample. i Q is the volume of the sample; n * Q represents the cumulative release rate per unit area at time point n. 投 This is the original dosage.

[0030] Plotting t as the x-axis, the cumulative in vitro release rate (Q) n * Plot tQ on the ordinate. n * The curve is obtained, and the curve is fitted to calculate the release equation.

[0031] As shown in Table 1 and Figure 10As shown in Table 2, the cumulative release rates of MTX and MTX-NS-loaded dual-network hydrogel microneedles within 72 h were 43.69±2.06% and 91.84±1.62%, respectively. At 72 h, the cumulative release rate of the MTX-NS-loaded dual-network hydrogel microneedles was 2.10 times that of MTX, indicating that the dual-network hydrogel microneedles improve the release rate of MTX and mitigate its slight water solubility. The in vitro cumulative release processes of both MTX and MTX-NS-loaded dual-network hydrogel microneedles conformed to a first-order kinetic model.

[0032] Table 1. Cumulative release rates of MTX and MTX-NS dual-network hydrogel microneedles (±s, n=3)

[0033] Table 2. In vitro cumulative release rate-time model fitting equations for MTX and MTX-NS dual-network hydrogel microneedles.

[0034] In vitro percutaneous permeation experiment of drug-loaded microneedles: The Franz diffusion cell method was used to conduct in vitro percutaneous permeation experiments on MTX active pharmaceutical ingredient (API), MTX-NS, and MTX-NS-loaded dual-network hydrogel microneedles. Skin was fixed between the receiving and supply cells, with the stratum corneum facing the supply cell and the dermis facing the receiving cell. 15 mL of pH 7.4 PBS solution containing 1% SDS was added to the receiving cell, ensuring close contact between the receiving solution and the skin, without air bubbles. 3.0 mL of MTX API, MTX-NS, and MTX-NS-loaded dual-network hydrogel microneedle solutions were added to the supply cells, respectively. The diffusion cell was placed in a constant temperature water bath at 37±0.5 ℃ with a stirring speed of 260 r·min. -1 Samples (3 mL) were taken at set times of 0.5, 1, 2, 3, 4, 6, 8, 10, 12, 24, 48, and 72 hours, with the same volume of fresh receiving solution added simultaneously. The sample solutions were filtered through a 0.22 mm microporous membrane, and the samples were analyzed using HPLC. The cumulative transdermal permeation rate (Q) for each group was calculated. n (mg.com) -2 ) and cumulative transdermal permeability Q n * (%), to examine the relationship between cumulative transdermal permeability and time (t, h). Cumulative transdermal permeability Q n (mg.com) -2 The calculation formula is (1-5); cumulative transdermal permeability Q n * The calculation formula for (%) is (1-6): ;

[0035] ; Among them: Q n C represents the cumulative transdermal permeability per unit area at time point n. n V represents the drug concentration measured at the nth time point, and V is the volume of the receiving liquid added. i Where A is the volume of each sample taken, Q is the effective transdermal area, and A is the effective transdermal area. n * Q represents the cumulative transdermal permeability per unit area at the nth time point. 投 This is the original dosage.

[0036] Plotting t as the x-axis, the cumulative transdermal permeability in vitro (Q) n * Plot tQ on the ordinate. n * The curve is obtained, and the curve is fitted to calculate the release equation.

[0037] As shown in Table 3 and Figure 11 As shown in Table 4, the cumulative percutaneous permeability of MTX within 72 h was 25.43±2.16%, that of MTX-NS within 72 h was 43.77±1.94%, and that of MTX-NS-loaded dual-network hydrogel microneedles within 72 h was 87.86±1.34%. The cumulative percutaneous permeability of MTX-NS-loaded dual-network hydrogel microneedles within 72 h was 3.10 times that of MTX, indicating that the dual-network hydrogel microneedles successfully penetrated the stratum corneum, increasing the permeability of MTX. The in vitro cumulative percutaneous permeability behavior of MTX, MTX-NS, and MTX-NS-loaded dual-network hydrogel microneedles all conformed to a first-order kinetic model.

[0038] Table 3. Cumulative transdermal permeability of MTX, MTX-NS, and MTX-NS-loaded dual-network hydrogel microneedles (±s, n=3)

[0039] Table 4. Fitting equations for the cumulative percutaneous permeability-time model of MTX, MTX-NS, and MTX-NS dual-network hydrogel microneedles.

[0040] The MTX-NS dual-network hydrogel microneedles prepared in this embodiment have an intact microneedle array, sharp tips, and good swelling properties. The hydrogel structure carrier release results show that the dual-network structure has a faster and longer drug release property than the single-network structure. Stability and swelling rate experiments show that the drug-loaded microneedles are relatively stable under relatively dry conditions and do not affect mechanical strength. Skin puncture experiments and rat skin histology results demonstrate that the MTX-NS dual-network hydrogel microneedles have good mechanical properties and form channels through swelling after entering the skin. Skin healing and skin safety evaluation experiments demonstrate that the MTX-NS dual-network hydrogel microneedles are non-irritating to the skin and have good safety. The rat skin swelling experiment of the MTX-NS dual-network hydrogel microneedles shows that the MTX-NS dual-network hydrogel microneedles are easy to peel off and not prone to breakage. In vitro release experiments... and After model fitting, the in vitro percutaneous transdermal perfusion experiments showed that the MTX-NS dual-network hydrogel microneedles all conformed to the first-order kinetic model.

[0041] Cell experiments Instruments: HBS-1096A ELISA reader - Nanjing Detie Experimental Equipment Co., Ltd.; LSM710 laser confocal microscope - Carl Zeiss, Germany; MCO-15AC CO2 incubator - SANYO, Japan. Materials: ADA (Catalog No.: A873854) was purchased from Shanghai Maclean Biotechnology Co., Ltd.; lipopolysaccharide (LPS, Catalog No.: L2880D) was purchased from Beijing Bio-Top Technology Co., Ltd.; fetal bovine serum (FBS, Catalog No.: A5256501) was purchased from Gibco; DMEM high glucose medium (Catalog No.: MA0212), penicillin-streptomycin sterile solution (Catalog No.: MA0110), cell cryopreservation solution (Catalog No.: MA0405), CCK-8 assay kit (Catalog No.: MA0225), live / dead cell staining assay kit (Catalog No.: MA0361), and reactive oxygen species (ROS) assay kit (Catalog No.: MA0219) were purchased from Dalian Meilun Biotechnology Co., Ltd.; NO ELISA kit (Catalog No.: E), TNF-α ELISA kit (Catalog No.: E), IL-10 ELISA kit (Catalog No.: E), and IL-1β ELISA kit (Catalog No.: E) were purchased from Andy Huatai Biotechnology Co., Ltd.

[0042] Animals: SPF-grade male SD rats, weighing 180-220 g, aged 6-7 weeks, purchased from Liaoning Growth Biotechnology Co., Ltd., production license SCXK (Liaoning) 2020-0001, animal experiment ethics committee number SYXK (Heilongjiang) 2021-018. Animal feeding and experimental procedures complied with the animal ethics guidelines of Jiamusi University.

[0043] Preparation of red blood cell suspension Take fresh rat blood into a centrifuge tube, add a small amount of physiological saline, shake well and wash, then centrifuge. Repeat the operation 3 times until the supernatant is no longer red. Collect the washed red blood cells, prepare a 2% red blood cell suspension with an appropriate amount of physiological saline, and store it in a 4℃ refrigerator for later use.

[0044] Hemolytic performance study 2% erythrocyte suspension was mixed with blank double-network hydrogel microneedles (B-HMN) and MTX-NS loaded double-network hydrogel microneedles (HMN), respectively. After 1 h of UV irradiation, the mixture was serially diluted to concentrations of 100, 75, 50, 25, 12.5, 6.25, and 3.125 mg / mL. -1 After gently shaking the suspension to mix it thoroughly, separate positive (0.5 mL 2% erythrocyte suspension + 0.5 mL deionized water) and negative (0.5 mL 2% erythrocyte suspension + 0.5 mL physiological saline) groups were set up. After incubation at 37℃ for 3 h, hemolysis was observed and photographed. The samples were centrifuged, and the absorbance of the supernatant was measured at 540 nm. The hemolysis rate of each group was calculated according to formula (1-7).

[0045] H 样 : Absorbance value of the experimental group samples; H 阳 : Absorbance value of the positive control group; H 阴 : Absorbance value of the negative control group.

[0046] like Figure 12 As shown, the B-HMN group and the HMN group at a concentration of 100 mg·mL -1 The hemolysis rates were 1.06% and 1.63%, respectively, far below the ISO 10993-5 safety standard (hemolysis rate <5%), demonstrating that the excipient carrier and drug formulation had low hemolytic effects on erythrocyte membranes. After centrifugation and ultraviolet irradiation, the hemolysis rates in each group increased slightly, but remained below 2%, indicating that the excipient carrier had good blood compatibility before and after ultraviolet irradiation, providing a safety basis for animal experimental drug administration using MTX-NS dual-network hydrogel microneedles.

[0047] RAW 264.7 cell culture RAW 264.7 cells are adherent cells, cultured in 1% penicillin-streptomycin double antibiotic, 10% fetal bovine serum and 89% DMEM high glucose medium, and in an incubator containing 5% CO2 and 37°C for subsequent experiments.

[0048] RAW 264.7 Cell Polarization RAW 264.7 cell polarization was induced using LPS, with 5×10⁻⁶ cells... 5 RAW 264.7 cells were seeded into 6-well plates and cultured overnight. When the cells reached 70% confluence, 2 mL of complete culture medium containing different concentrations of inducing factors was added: one for normal cells and one for cells containing 50 ng / mL of inducing factors. -1 LPS, 100 ng·mL -1 LPS, 400 ng·mL -1 LPS, 700 ng·mL -1 LPS, 1 mg·mL -1 LPS, 1.2 mg·mL -1 LPS was used in three replicates per group. After co-culturing with cells for 24 h and 48 h, cell morphology was observed and the supernatant from each well was collected. The NO content was measured according to the kit to determine whether the cells were successfully polarized.

[0049] RAW 264.7 cells treated with different concentrations of LPS were observed for cell morphology at 24 h and 48 h, respectively. Figure 13 As shown, before polarization, the cells were round; after polarization, the cell morphology became irregular, forming multiple pseudopodia, and the volume increased significantly. The NO concentration in the cell supernatant after polarization is shown in the following figures. Figure 17 As shown, the amount of NO secreted by RAW 264.7 cells increased with increasing LPS treatment time, and the amount of NO secreted by RAW 264.7 cells increased when the LPS concentration was 100 ng·mL⁻¹. -1 When the treatment time was 48 h, the amount of NO secreted by the cells was significantly higher than that of other LPS concentrations. Therefore, the polarization conditions were set at an LPS concentration of 100 ng·mL. -1 The polarization time was 48 h, which was used as a method for establishing inflammatory cell models, such as... Figure 14 As shown.

[0050] Cytotoxicity studies 1×10 5 RAW 264.7 cells were seeded in 96-well plates. After 24 hours, the culture medium was discarded, and serially diluted sample solutions were added. Experiments were then conducted on polarized RAW 264.7 cells and normal RAW 264.7 cells, respectively. Specific experimental groups are shown in Table 5.

[0051] Table 5. Results of Cytotoxicity Studies (Grouped)

[0052] Each group was configured with three replicates. After culturing for 48 h, 10 mL of a cytotoxicity assay kit (CCK-8) was added to each well, and the cells were incubated for another 1 h. The absorbance (A) of each well was measured at 450 nm. Cell viability was calculated using the following formula (1-8):

[0053] Where A 样本 A 空白 and A 对照 These represent the sample solution group, blank group, and control group, respectively.

[0054] like Figure 15 As shown, the B-HMN group, as a carrier material, had no significant toxic side effects on both normal and polarized RAW 264.7 cells, demonstrating good biocompatibility. The MTX and HMN groups ranged from 4.54 to 13.63 mg / mL. -1 Within the concentration range, with increasing MTX concentration, the viability of normal and polarized RAW 264.7 cells remained above 80%, while the viability of the MTX and HMN groups ranged from 18.18 to 22.72 mg / mL. -1 Within the concentration range, as the MTX concentration increased, the viability of normal and polarized RAW 264.7 cells decreased to below 80%.

[0055] In the combined group, when the ADA concentration was 0.37 mg·mL -1 ~0.74 mg·mL -1 Within a certain concentration range, cell viability gradually decreased with increasing MTX concentration, but remained above 80%, demonstrating good biocompatibility. When the ADA concentration was 7.4 mg / mL... -1 At the same time, with increasing MTX concentration, cell viability before and after polarization decreased to below 80%. Based on the above experimental results and the clinical dosage of ADA, CCK-8 determined the final concentration for each experimental group in conjunction with the clinical dosage of adalimumab for subsequent animal experiments. The B-HMN group was 1.46 mg / mL. -1 ~7.3 mg·mL -1 The HMN and MTX groups had a concentration of 4.54 mg / mL. -1 ~13.63 mg·mL -1 The ADA solution concentration was 0.74 mg / mL. -1 .

[0056] Cell live-death experiment Take polarization 2×10 5 RAW 264.7 cells were seeded into 12-well plates and cultured overnight. Complete culture medium containing PBS was then added to each well. The HMN group (containing 4.54 mg / mL MTX) was also included. -1 9.09 mg·mL -1 13.63 mg·mL -1 Complete culture medium; combined group (containing 4.54 mg / mL MTX) -1 9.09 mg·mL -113.63 mg·mL -1 ADA solution 0.74 mg·mL -1 Sixteen groups were subjected to RAW 264.7 cytotoxicity experiments using complete culture medium.

[0057] Each group was set up with three replicates. After culturing for 24 h, 100 mL of staining working solution was added to each well according to the Calcein AM kit requirements. The cells were incubated in a cell culture incubator for 15-30 min, with 2-3 washes during this period, each wash lasting less than 1 min. The cell state was observed and photographed under a laser confocal microscope (CLSM) at 515 nm. The images were then semi-quantitatively processed using ImageJ.

[0058] The fluorescent staining agent AM can stain live cells green, and the results of cell viability experiments can be analyzed by green fluorescence colorimetric analysis, such as... Figure 16 and Figure 17 As shown, the PBS group exhibited more green fluorescence, indicating a large number of surviving cells, with a cell survival rate of 97%, demonstrating high biosafety. In the HMN group, green fluorescence decreased with increasing concentration, and the cell survival rate was 89%. In the combined group, the number of dead cells increased significantly, and the cell survival rate was the lowest at 84% with increasing MTX concentration, indicating that the combined treatment was more effective than the microneedle therapy group. This result is consistent with the cytotoxicity experiment results.

[0059] Cellular anti-inflammatory effects Take polarized RAW 264.7 cells and add 1×10 5 RAW 264.7 cells were seeded into 12-well plates. Experiments were conducted when the RAW 264.7 cells reached 85% confluence, divided into a control group (normal cells), a model group (polarized cells), and an HMN group (4.54 mg / mL). -1 9.09 mg·mL -1 13.63 mg·mL -1 ), combined group (containing 4.54 mg / mL of MTX) -1 9.09 mg·mL -1 13.63 mg·mL -1 ADA solution 0.74 mg·mL -1 Each group was set up with three replicates. After 48 h of incubation, the supernatant from each well was collected, and the levels of released NO, TNF-α, IL-10, and IL-1β were detected according to the kit's operating procedure.

[0060] IL-10 is an anti-inflammatory factor, while TNF-α and IL-1β are pro-inflammatory factors. NO content is an important indicator of successful polarization in RAW 264.7 cells. (See Table 8 and...) Figure 18As shown in Figure a, the comparison between the blank group and the model group P <0.001, indicating a highly significant difference, demonstrates an abnormally low level of IL-10 anti-inflammatory factor in the model group, indicating the successful establishment of the inflammation model; comparison between the model group and the HMN group. P <0.001, indicating a highly significant difference, demonstrating that the HMN group can promote the upregulation of IL-10 anti-inflammatory factors; comparison between the HMN group (high, medium, and low doses) and the positive control group. P <0.001, the HMN group showed a better ability to upregulate IL-10 at all concentrations than the positive group; compared with the combined group, the positive group showed a significantly higher ability to upregulate IL-10. P The difference was <0.001, indicating a highly significant difference. This suggests that the combined group had a better ability to upregulate IL-10 anti-inflammatory factors than the positive group. These results indicate that both the HMN (high, medium, and low doses) group and the combined group had a strong ability to upregulate IL-10 anti-inflammatory factors, and the combined group had a better upregulation ability than the HMN group.

[0061] As shown in Table 8 and Figure 18 As shown in b, the model group is compared with the HMN group (high, medium, and low doses) and the combination group. P <0.001, indicating a highly significant difference, demonstrating that both the HMN group and the combination group can promote the downregulation of IL-1β pro-inflammatory factors, and the downregulation ability of both the HMN group and the combination group is superior to that of the positive group; comparison between the HMN (high, medium, and low dose) group and the combination group. P The difference was <0.001, indicating a highly significant difference, demonstrating that the combined group had a better ability to downregulate IL-1b pro-inflammatory factors than the HMN group. These results indicate that both the HMN (high, medium, and low doses) group and the combined group had a strong ability to downregulate IL-1b pro-inflammatory factors, and the combined group had a better downregulation ability than the HMN group.

[0062] As shown in Table 8 and Figure 18 As shown in Figure c, the model group is compared with the HMN group (high, medium, and low doses) and the combination group. P <0.001, indicating a highly significant difference, demonstrating that both the HMN group and the combination group can promote the downregulation of TNF-α pro-inflammatory factors, and the downregulation ability of both the HMN group and the combination group is superior to that of the positive group; comparison between the HMN (high, medium, and low dose) group and the combination group. P The difference was <0.001, indicating a highly significant difference, demonstrating that the combined group had a better ability to downregulate TNF-α pro-inflammatory factor than the HMN group. These results indicate that both the HMN (high, medium, and low doses) group and the combined group had a strong ability to downregulate TNF-α pro-inflammatory factor, and the combined group had a better downregulation ability than the HMN group.

[0063] As shown in Table 6 and Figure 18 As shown in d, the model group is compared with the HMN group (high, medium, and low doses). P <0.001, indicating a highly significant difference, demonstrating that the HMN group can promote NO downregulation, and the downregulation ability of the HMN group is superior to that of the positive control group; comparison between the HMN group (high, medium, and low doses) and the positive control group.P <0.001, the ability of the HMN group to downregulate NO at all concentrations was superior to that of the positive control group; comparison between the HMN (high, medium, and low doses) group and the combined group. P The difference was <0.001, indicating a highly significant difference, demonstrating that the combined group had a better ability to downregulate NO than the HMN group. These results show that both the HMN (high, medium, and low doses) group and the combined group had a strong ability to downregulate NO, and the combined group had a better ability to downregulate NO than the HMN group.

[0064] In summary, the MTX-NS dual-network hydrogel microneedles exhibit good anti-inflammatory capabilities, and their effects are even more pronounced when used in combination with ADA, providing a basis for subsequent animal experiments.

[0065] Table 6. Results of LPS-induced inflammatory factor content detection in RAW 264.7 cells in each group (±s, n=3)

[0066] Cell Western Blot Analysis 4×10 5 RAW 264.7 cells were seeded into 6-well plates. The experiment was divided into 5 groups: the control group received no treatment; the model group was treated with LPS to establish an RA inflammatory cell model; and the positive group was treated with LPS for 2 hours and then given a solution containing 7.31 mg / mL of LPS. -1 The positive control group was treated with diclofenac sodium cream solution; the HMN group was given a solution containing 13.632 mg / mL after 2 h of LPS treatment. -1 The intervention was performed using a solution of MTX-loaded dual-network hydrogel microneedles; the combined group was given a solution containing 13.632 mg / mL of MTX after 2 h of LPS treatment and incubation. -1 MTX drug-loaded microneedle solution and 0.74 mg / mL -1 Intervention was performed using ADA solution.

[0067] Each group was configured with three replicates. Protein was extracted after 24 hours using inducible iNOS and COX-2 total protein extraction kits. Protein concentration was determined using a BCA assay kit. Proteins were denatured by heating in a water bath at 100°C for 10 minutes, aliquoted, and stored at -80°C for later use. Separating and stacking gels were prepared separately, and protein samples were loaded onto them. Total protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (room temperature, 120 kV, 100 min). After transfer at 300 mA on ice for 1.5 hours, the samples were transferred to a polyvinylidene fluoride membrane, blocked in 5% skim milk for 1 hour, washed with TBST for 1 minute, and incubated overnight at 4°C with primary antibody (iNOS and COX-2, both diluted 1:1000). Wash the membrane with TBST (5 times, 5 min each time), add secondary antibody, incubate at room temperature for 1 h, wash the membrane again with TBST, add developing solution and place it in a chemiluminescent gel imaging system for imaging, analyze the gray values ​​using ImageJ software, and perform relative quantitative analysis.

[0068] Activation of the iNOS protein promotes the release of NO, and NO, a pro-inflammatory mediator, stimulates the release of pro-inflammatory factors such as TNF-α, IL-1β, and IL-10. The catalytic products of the COX-2 protein also promote the release of NO and pro-inflammatory factors.

[80] Elevated levels of NO and pro-inflammatory factors in the body will lead to tissue DNA damage, promote deeper inflammation and tissue edema, and accelerate the development of inflammatory diseases.

[0069] like Figure 19 and 20 As shown, the model group is compared with the blank group. P <0.01 indicates a significant difference, proving the successful establishment of the inflammation model; the positive group and the microneedle group were compared with the model group respectively. P <0.01 indicates a significant difference, demonstrating that both the positive control group and the microneedle group can downregulate the expression of iNOS and COX-2 proteins, with the microneedle group showing a superior ability to downregulate iNOS protein compared to the positive control group; the microneedle group was compared with the combined drug administration group. P The difference was <0.01, indicating a significant difference, suggesting that the combined administration group had a better ability to downregulate the expression of iNOS and COX-2 proteins than the positive control group. This further supports the good therapeutic effect of MTX-NS dual-network hydrogel microneedles and ADA in treating inflammation.

[0070] Study on the therapeutic effect of MTX-NS dual-network hydrogel microparticles on RA model rats An AA rat model was established, and the model rats were treated with medication. The success of the model and the evaluation of the treatment intervention were determined by rat weight, rat toe arthritis score, rat foot volume, and rat paw thickness. After the treatment ended on day 49, X-ray imaging, ankle joint histopathological section, and immunohistochemical evaluation were performed to verify the therapeutic efficacy of MTX-NS dual-network hydrogel microneedles and the feasibility of combined use with ADA based on the level of inflammation and the angle of cartilage loss in vivo.

[0071] Instruments: PDMS mold (needle height: 800 m) Micropoint Technologies PTE LTD, TDZ5-WS benchtop low-speed centrifuge - Hunan Xiangyi Laboratory Instrument Development Co., Ltd., FA2004N electronic analytical balance - Shanghai Shunyu Hengping Scientific Instrument Co., Ltd., DF-101S thermostatic heating magnetic stirrer - Gongyi Yuhua Instrument Co., Ltd., HAD-LS7C toe volume measuring instrument - Beijing Heng'aode Instrument Co., Ltd., vernier caliper - Anhui Sandu Duzhi Cutting Tool Co., Ltd., disposable syringe - Shandong Xinhua Ande Medical Supplies Co., Ltd., pressure-sensitive tape - Jinhua Jingdi Medical Supplies Co., Ltd.

[0072] Materials: Freund's complete adjuvant (drug code: F850325) and MTX (hydrate, 99% purity, drug code: M813626) were purchased from Shanghai Maclean Biochemical Technology Co., Ltd., ADA (drug code: A412002) was purchased from Beijing Huayuan Interconnect Technology Co., Ltd., sodium pentobarbital (drug code: BC1040) was purchased from Beijing Pruton Biotechnology Co., Ltd., diclofenac sodium cream was purchased from Suzhou Yifan Pharmaceutical Co., Ltd., 75% ethanol was purchased from Nanyang Huikang Biotechnology Co., Ltd., physiological saline was purchased from Heilongjiang Qitai Animal Health Products Co., Ltd., 4% paraformaldehyde fixative was purchased from Fuzhou Xijing Biotechnology Co., Ltd., IL-6 immunohistochemistry kit was purchased from Shanghai Jingfeng Biotechnology Co., Ltd., and TNF-α and IL-1β immunohistochemistry kits were purchased from Shanghai Yaji Biotechnology Co., Ltd.

[0073] Animals: SPF-grade male SD rats, weighing 180-220 g and aged 6-7 weeks, were purchased from Liaoning Growth Biotechnology Co., Ltd., production license number SCXK(Liaoning) 2020-0001, animal experiment ethics committee number SYXK(Heilongjiang) 2021-018. Animal husbandry and experimental procedures complied with the animal ethics guidelines of Jiamusi University. The laboratory was maintained at a temperature of 25±0.5℃, relative humidity of 40%–60%, alternating light and dark cycles, and free access to food and water. The rats underwent acclimatization training for one week prior to the experiment.

[0074] Construction of AA rat model The left paw pad of the rat was disinfected with 75% ethanol, and 0.1 mL of Freund's complete adjuvant was injected subcutaneously into the paw pad for the first immunization. On day 8, a second 0.1 mL of Freund's complete adjuvant was injected into the same site to boost the immunization, thus successfully establishing the AA rat model.

[0075] Experimental animal grouping and dosing regimen Treatment began 15 days after the second immunization of rats. The AA model rats were randomly divided into six groups: blank group (no modeling rats) n6, model group n6, positive group n6, HMN group n6, ADA group n6, and combined group n6, for a total of 36 rats.

[0076] Following the clinical protocol for treating rheumatoid arthritis with MTX and ADA, the drugs were administered once a week for a total of 5 times. Saline was applied to the feet of the model group and the control group; 0.05 g of diclofenac sodium cream was applied to the ankle joint of the positive control group; in the HMN group, drug-loaded microneedles were inserted into the dorsum of the rat's foot and secured with pressure-sensitive tape; the ADA group received a subcutaneous injection of 1.2 mg / mL. -1 ADA solution 0.3 mL; the combined group repeated the operation of the microneedling group and the ADA group. To avoid repeated operation, the ADA solution was injected 4 h before the microneedling transdermal drug delivery was performed.

[0077] Rat paw arthritis score According to the standards of the European League Against Rheumatism and the American College of Rheumatology (EULAR / ACR), the paw scores of rats were assessed every 4 days after administration on day 8. Each paw was scored from 0 to 4 points, and the theoretical maximum score for arthritis in each rat was 16 points. The scoring criteria are shown in Table 7 below:

[0078]

[0079] like Figure 21 As shown, the arthritis scores of rats in the blank control group were all 0; the arthritis scores of rats in the model group continued to increase from day 8 to day 40, and only slightly decreased after day 40; compared with the model group, the scores of rats in the positive control group gradually increased from day 8 to day 28, and began to decrease from day 28, indicating that the swelling of the rat arthritis gradually began to improve; the scores of the HMN group began to decrease after day 24, indicating that the inflammation gradually improved; the scores of the ADA group began to decrease from day 20; and the scores of the combined group began to decrease from day 16. In summary, compared with the model group, during the treatment period from day 8 to day 48, the scores of each treatment group showed improvement. P The difference was <0.001, indicating a significant difference. This suggests that all treatment groups could improve the swelling of arthritis in rats, and the improvement scores of the HMN group and the combined group were better than those of the positive group.

[0080] Rat paw thickness measurement The thickness of the rat's paw was measured using calipers to assess the swelling of the rat's paw. Starting from day 8, the middle of the left paw of the rat was measured every 4 days, and the data were recorded to examine whether the model was successfully established and the drug treatment effect.

[0081] Visual image of rat paw swelling after treatment (see image below) Figure 22 As shown, in the control group, rats had no swelling in their feet, which were relatively flat, with obvious joints in the toes; in the model group, rats had severe foot swelling, with rounded swollen toes, indistinct joints in the toes, and significant swelling in the ankle joints; in the positive group, compared to the model group, the toe joints showed slight improvement, and foot swelling was reduced; in the HMN group, swelling in the soles and dorsum of the feet was significantly reduced, joints in the toes were slightly more prominent, and there was relief in the ankle joints; the ADA group showed improvement in foot swelling similar to the microneedling group; in the combined group, foot swelling was significantly reduced, and joints in the toes were slightly more prominent. In summary, all treatment groups showed significant improvement in relieving foot swelling in rats.

[0082] Results of rat paw drainage volume as follows Figure 23 As shown, the foot volume of rats in the blank control group remained relatively consistent at all treatment time points; the foot volume of rats in the model group increased slowly from day 8 to day 40, and decreased slightly after day 40, possibly due to autoimmune effects and slight relief of inflammation; the foot volume of rats in the positive control group began to decrease on day 36, and inflammation was reduced; the foot volume of rats in the HMN group began to decrease on day 24, showing better results than the positive control group; the foot volume of rats in both the ADA group and the combined group began to decrease on day 20, with the combined group showing a larger decrease and faster recovery than the ADA group alone. This demonstrates that both the HMN group and the combined group can effectively promote a decrease in foot volume in rats.

[0083] The results of rat paw thickness measurement are as follows Figure 24 As shown, the thickness of each paw pad in the blank group remained relatively consistent during treatment; the paw pad thickness in the model group slowly increased from day 8, and decreased slightly on day 44, possibly due to autoimmune effects and a slight reduction in inflammation; the paw pad thickness in the positive group began to decrease on day 36, indicating a reduction in inflammation; the paw pad thickness in the HMN group began to decrease on day 24, with a greater degree of decrease than in the positive group; both the ADA group and the combined group saw a decrease in paw pad thickness starting on day 20, with the combined group showing a larger decrease and faster recovery than the ADA group alone. In summary, compared to the model group, each treatment group significantly reduced paw pad thickness in rats. P <0.001 indicates a significant difference, meaning that each group showed significant improvement.

[0084] X-ray imaging examination On day 49 of the experiment, rats were anesthetized with 2% sodium pentobarbital, and their left ankle joints were examined using an X-ray imaging system.

[0085] X-ray results of rat paws as follows Figure 25As shown, the blank group rats had no foot swelling and the bones were intact and undamaged; the model group rats had obvious foot swelling, reduced joint spaces, bone erosion and hyperplasia, and slight stiffness in the limbs; the positive group rats had enlarged joint spaces in the feet and the foot swelling was still obvious; the ADA group and HMN group rats showed further reduction in foot swelling compared to the model group, and the joint spaces were clear; the combined group rats showed significant reduction in foot swelling and clear, intact joints, which was consistent with the results of rat toe thickness and toe score.

[0086] Histopathological analysis of foot and ankle joint After treatment on day 49, rats were euthanized by intraperitoneal injection of an excessive amount of 2% sodium pentobarbital. The intact ankle joints of the rats were removed, and the skin, flesh, and hair were stripped off. The rats were fixed in 4% paraformaldehyde, decalcified in 10% EDTA, embedded in paraffin, and sectioned. Histopathological analysis was performed using both hematoxylin-eosin (H&E) and safranin-fast green staining methods.

[0087] Histopathological results of rat paw joint tissues as follows Figure 26 As shown, the model group clearly exhibited extensive inflammatory cell infiltration and bone erosion symptoms, with significant synovial hyperplasia. Compared to the model group, the positive control group showed slight relief, but inflammatory cell infiltration and bone erosion symptoms remained pronounced. The HMN group showed a reduction in inflammatory cell infiltration and bone erosion symptoms, with narrowing of the joint cavity and improvement in synovial hyperplasia. The pathological results of the ADA group were similar to those of the microneedle group. The combined treatment group showed significant improvement in inflammatory cell infiltration and bone erosion symptoms, with larger and wider joint cavity openings, and effective improvement in the joint pathological state. In summary, compared with the model group, all treatment groups significantly alleviated the inflammatory pathological changes in rats.

[0088] Cartilage tissue is composed of chondrocytes, cartilage matrix, and fibers. The principle of Safranin-Fix Green staining is that basophilic cartilage tissue combines with the basic dye Safranin to produce a red stain, while eosinophilic bone tissue combines with the acidic dye Fast Green to produce a blue or green stain, thus distinguishing cartilage from bone tissue. If the cartilage tissue is damaged or unevenly distributed, the Safranin staining will be lighter or uneven. Figure 27 As shown, in the blank group, the ankle cartilage of rats was clearly stained and intact; in the model group, the joint cavity of rats was narrowed, the degree of cartilage staining was low, and the damaged area was large; in the positive group, the degree of cartilage damage was improved to some extent, but the joint cavity was still narrow; in the HMN group, the joint cavity of rats was widened, and the degree of inflammation of cartilage attachment was obviously improved on the joint surface; the inflammation improvement in the ADA group was the same as that in the microneedle group; in the combined group, the cartilage attachment position was further increased, the degree of cartilage damage was better improved, the joint cavity gap began to widen to a large extent, and the cartilage surface was relatively smooth.

[0089] Both H&E and Safranin-Fix Green staining results demonstrated that both the HMN group and the combined group had a good effect on improving rheumatoid arthritis inflammation, and the combined group was superior to the HMN group.

[0090] Immunohistochemical analysis of ankle joint The rats underwent the same treatment. After embedding the ankle joint sections, the knee and ankle joint tissue sections were subjected to antigen retrieval for 23 min in a microwave oven using EDTA antigen retrieval buffer. Then, 3% H2O2 solution was added and the sections were treated for 25 min in the dark to block endogenous peroxidase. The sections were blocked with 3% BSA in PBS solution for 30 min, followed by incubation overnight at 4°C with primary antibodies (anti-IL-6, anti-IL-1b, and anti-TNF-a). HRP-labeled secondary antibody was then added and incubated at room temperature for 1 h. DAB chromogenic solution was added for staining; positive results showed a brownish-yellow color. Hematoxylin was used for counterstaining for 3 min. The sections were then mounted with neutral resin after dehydration. The sections were photographed using an Olympus fluorescence microscope, and semi-quantitative analysis was performed using Image Pro Plus 6.0 image analysis software.

[0091] Bone injury results from abnormal osteoclast proliferation and osteoblast inhibition, and is a major pathological feature of rheumatoid arthritis. TNF-α induces abnormal activation of osteoclasts, stimulates osteoclast formation, and induces osteoblast apoptosis. IL-1β inhibits osteoblast proliferation and migration, and synergistically promotes osteoclast differentiation with TNF-α. IL-6 similarly promotes osteoclast differentiation and inhibits osteoblast differentiation.

[0092] Immunohistochemical results of rat ankle joints as follows Figure 28 and Figure 29 As shown, in the semi-quantitative analysis of the inflammatory factor IL-1β, the model group was compared with the blank group. P The difference was <0.001, indicating a highly significant difference. Furthermore, the model group showed a large number of brownish-yellow positive areas representing inflammation, confirming the successful establishment of the AA rat model. The model group was compared with the HMN group and the combined group, respectively. P <0.001, indicating a highly significant difference, and a significant reduction in the inflammatory brown-yellow area in both the HMN group and the combined group, demonstrating that both the HMN group and the combined group can promote the reduction of the inflammatory factor IL-1b; the positive group compared with the HMN group and the combined group P <0.001, indicating a highly significant difference. The positive group had slightly more brownish-yellow areas than the microneedle group and the combined drug administration group, indicating that the HMN group and the combined drug administration group were superior to the positive group in reducing IL-1b expression.

[0093] In the expression of the inflammatory cytokine IL-6, the model group was compared with the blank group. P<0.001, indicating a highly significant difference; the model group showed numerous inflammatory brown-yellow positive areas, proving the successful establishment of the AA rat model; the model group was compared with the HMN group and the combined group, respectively. P <0.001, indicating a highly significant difference, showed a significant reduction in brownish-yellow inflammatory areas in both the HMN group and the combined group, demonstrating that the HMN group and the combined group can promote the reduction of the inflammatory factor IL-6; the positive group was compared with the HMN group and the combined group. P <0.001, indicating a highly significant difference. The positive group showed slightly more brownish-yellow areas than the HMN group and the combined group, suggesting that both groups were superior to the positive group in reducing IL-6 expression. Comparison between the HMN group and the combined group... P <0.001, the combined group had fewer brownish-yellow inflammatory areas, and the combined ADA intervention on the basis of the same drug concentration microneedles was better than the HMN group, indicating that the drug-loaded microneedles still have a strong ability to reduce IL-6 expression after combined administration.

[0094] In the expression of the inflammatory factor TNF-α, the model group was compared with the blank group. P <0.001, indicating a highly significant difference; the model group showed a large number of brownish-yellow positive areas representing inflammation, proving the successful establishment of the AA rat model; the model group was compared with the HMN group and the combined group, respectively. P <0.001, indicating a highly significant difference, showed a significant reduction in brownish-yellow inflammatory areas in both the HMN group and the combined group, demonstrating that the HMN group and the combined group can promote the reduction of the inflammatory factor TNF-α; the positive group was compared with the HMN group and the combined group. P The value <0.001 indicates a highly significant difference. The positive group had slightly more brownish-yellow areas than the microneedle group and the combined group, indicating that the two groups were superior to the positive group in reducing TNF-α expression.

[0095] This study investigated the effect of MTX-NS dual-network hydrogel microneedles on rheumatoid arthritis (RA) inflammation by establishing an AA rat model. The model was evaluated using rat body weight, toe arthritis scores, foot volume, and foot thickness measurements. The therapeutic effect of MTX-NS dual-network hydrogel microneedles was verified from an in vivo inflammatory perspective using X-ray imaging, ankle joint histopathological sections, and immunohistochemistry. Compared to clinically used diclofenac sodium cream, the drug-loaded microneedles alleviated the progression of RA and reduced inflammation in the joints. This chapter also verified that the treatment results of the microneedle combined with ADA injection regimen were superior to the microneedle group. This administration method not only follows the clinical treatment regimen of combining MTX and ADA but also provides experimental evidence for the improvement and upgrading of new MTX formulations.

[0096] 1. The optimal formulation for dual-network hydrogel microneedles, optimized using BBD, is M. mHA :M GelMA The ratio is 1:2, M mHA :MGelMA :M PVP K30 The ratio is 1:1:5, M (mHA+GelMA) :M 水 The ratio is 1:12; the optimal preparation conditions are a centrifugation speed of 3120 r·min. -1 The first UV crosslinking time was 86 min, and the second UV crosslinking time was 187 min. The prepared double-network hydrogel microneedles exhibited complete needle emergence and good swelling capacity, and were successfully applied in Parafilm. ® The membrane has a puncture rate of approximately 98% and a bending resistance of approximately 9.7%, exhibiting good mechanical properties.

[0097] 2. A UV full-wavelength scan was used to select 302 nm as the detection wavelength for MTX. The in vitro analytical method for MTX showed good linearity using HPLC. MTX-NS was prepared using a solvent-nonsolvent precipitation method, with a particle size of 325.72 ± 5.7 nm, a PDI of 0.154 ± 0.16, and a potential of -11.4 ± 1.2 mV. FTIR and DSC both confirmed the successful preparation of MTX-NS. The drug loading of MTX-NS was 61.3%, and the cumulative release rate of MTX-NS at 72 h was 1.59 times that of MTX, indicating that MTX-NS improved the release rate of MTX and mitigated the poor solubility of MTX.

[0098] 3. MTX-NS-loaded dual-network hydrogel microneedles were prepared using a centrifugation-molding method. The microneedle array was intact, with sharp tips and good swelling properties. The needles completely disappeared within 12 minutes, exhibiting faster and longer drug release characteristics compared to single-network microneedles. The structure remained stable after drug loading, demonstrating good mechanical, swelling, and safety properties, and was easily peeled off without easily breaking. DSC and FTIR studies confirmed the successful encapsulation of MTX-NS within the dual-network hydrogel microneedles. The in vitro cumulative percutaneous permeability behavior of the MTX-NS-loaded dual-network hydrogel microneedles conformed to a first-order kinetic model.

[0099] 4. The microneedle carrier excipient has no hemolytic effect; the polarizing concentration for RAW 264.7 cells is 100 ng / mL. -1 The polarization time was 48 h. After successful induction, the cells exhibited irregular morphology, multiple pseudopodia, and increased volume. Cytotoxicity and cell viability assays determined safer concentration ranges for each experimental group. Cell scratch assays showed that the MTX-NS dual-network hydrogel microneedles were superior to the positive control group, and the effect was even better when combined with ADA, with the inhibition of cell migration showing a concentration-dependent effect. In in vitro cell experiments, both the drug-loaded microneedles and the combination group exerted anti-inflammatory effects by significantly upregulating the anti-inflammatory factor IL-10 and downregulating pro-inflammatory factors TNF-α, IL-1β, NO, and inflammation-related proteins iNOS and COX-2.

[0100] The use of MTX-NS dual-network hydrogel microneedles in in vivo animal models of inflammation treatment has demonstrated that the microneedle group and the combined group are effective in improving joint swelling, reducing bone erosion and histopathological damage. The system has also verified the synergistic therapeutic effect of drug-loaded microneedles combined with ADA on RA, providing a painless, efficient and precise new strategy for RA treatment.

Claims

1. A method for preparing a dual-network hydrogel microneedle, characterized in that, The method includes the following steps: Step 1: Dissolve methotrexate (MTX) in NaHCO3 solution, then add anhydrous ethanol and mix well to obtain solution A; Step 2: Dissolve PVP K30 in purified water to obtain solution B; Step 3: At 35°C, while continuously stirring, inject solution A into solution B, and continue stirring to obtain MTX-NS for later use; Step 4: Weigh out PVP K30 and MTX-NS, mix them, and place them in a 4°C refrigerator to swell. Add hyaluronic acid methacrylate HAMA, methacrylamide gelatin GelMA and photoinitiator I2959, and place them in a 35°C water bath and stir continuously until the solution is evenly mixed. Let them swell at room temperature to obtain a pregel solution. Step 5: After casting the pregel solution onto the microneedle mold, centrifuge it, then irradiate it with ultraviolet light, pour in the same pregel solution again, centrifuge it again to fully fill the mold backing area, and then irradiate it with ultraviolet light to obtain the MTX-NS dual-network hydrogel microneedles.

2. The method according to claim 1, characterized in that, In step three, when solution A is injected into solution B for mixing, the volume ratio between the two is 1:7, the volume ratio of MTX to PVP K30 is 2:3, and the stirring time is 62 min.

3. The method according to claim 1, characterized in that, In step three, a centrifuge is used for stirring at a speed of 3120 r·min. -1， Centrifugation time was 10 min.

4. The method according to claim 1, characterized in that, In step four, the mass ratio of methacrylamide hyaluronic acid (mHA) to methacrylamide gelatin (GelMA) is 1:

2.

5. The method according to claim 1, characterized in that, In step four, the mass ratio of methacrylamide hyaluronic acid (mHA) to polyvinylpyrrolidone K30 (PVP K30) is 1:

5.

6. The method according to claim 1, characterized in that, In step four, the mass ratio of methacrylated hyaluronic acid to water is 1:

36.

7. The method according to claim 1, characterized in that, In step five, the centrifugation speed was 3120 r·min for both times. -1 The first UV crosslinking time was 86 min, and the second UV crosslinking time was 187 min.

8. MTX-NS dual-network hydrogel microneedles are prepared by any of the preparation methods described in claims 1-9.

9. Use of the microneedle gel according to any one of claims 1 to 9 in the manufacture of a product for the in vivo anti-inflammatory effect of ADA.

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