PH-responsive hydrogel microneedle as well as preparation method and application thereof

Abstract

The invention discloses a pH response hydrogel microneedle as well as a preparation method and application thereof. The pH response hydrogel microneedle comprises a hydrogel microneedle main body and colchicine loaded in the hydrogel microneedle main body, the hydrogel microneedle main body is hydrogel with a three-dimensional network structure, which is formed by covalent cross-linking of methacrylated hyaluronic acid and methacrylated orthoester. The hydrogel microneedle prepared by taking the methacrylated hyaluronic acid and the methacrylated orthoester as cross-linking components not only has good mechanical property, swelling property and biocompatibility, but also can respond to the microenvironment pH of an inflammation part by virtue of the pH sensitivity of the methacrylated orthoester, so that the utilization rate of a medicine is improved, and the bioavailability of the medicine is improved. The acute gout arthritis can be quickly and efficiently treated, and the clinical application potential is huge.

Description

Technical Field

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

[0002] Gouty arthritis is an inflammatory reaction caused by the deposition of sodium urate crystals (MSU) within and around joint structures. Recent epidemiological studies show an increasing prevalence and incidence of acute gout. Acute gout attacks occur suddenly, causing severe pain and limiting mobility. In severe cases, it can lead to joint deformities and related joint damage, causing significant suffering and burden on patients. Colchicine (Col) is the first-line anti-inflammatory drug for the clinical treatment of acute gout, effectively relieving the pain and swelling caused by acute attacks. However, Colchicine has a narrow therapeutic window, and common clinical side effects include gastrointestinal toxicity, causing diarrhea, nausea, vomiting, and stomach discomfort. Therefore, minimizing its side effects is crucial when using Colchicine to treat acute gout.

[0003] Microneedles (MNs) are a novel drug delivery system that offers unique advantages over oral and subcutaneous injections, including painlessness, better patient compliance, fewer side effects, and high transdermal delivery efficiency. Due to the wide selection of materials, high processability, and ideal biocompatibility, next-generation smart microneedles are typically polymer microneedles, including soluble microneedles and hydrogel microneedles. Studies have shown that delivery of Col via soluble microneedles results in 3.36 times higher in vitro transdermal absorption compared to gel delivery, with moderate relative bioavailability in vivo, significantly reducing knee swelling and mechanical sensory impairment in a rat model of acute gout. Although soluble microneedles can rapidly release drugs, they still cannot avoid toxic drug bursts similar to those seen with Col, leading to excessively high local drug concentrations and potential drug toxicity hazards.

[0004] Chinese patent application CN202211077519.0 discloses a method for preparing colchicine hydrogel microneedles, which mainly controls drug release through morphological changes in the cross-linked hydrogel network structure. Its significant advantage lies in controlling the drug release rate and the mechanical strength of the microneedles by controlling the degree of cross-linking of the hydrogel. However, to ensure sufficient mechanical strength for the microneedles to sting the skin, a high degree of cross-linking and a dense network structure are required, restricting drug escape from the network structure. This is not conducive to the rapid anti-inflammatory and analgesic effect in acute gout attacks.

[0005] Therefore, researching a hydrogel microneedle that can achieve rapid anti-inflammatory and analgesic effects while maintaining sufficient mechanical strength to pierce the skin has good clinical application value. Summary of the Invention

[0006] To address the shortcomings of the prior art, the present invention aims to provide a pH-responsive hydrogel microneedle that not only possesses excellent mechanical properties, swelling properties, and biocompatibility, but also responds to the pH of the microenvironment at the disease site, significantly improving drug release efficiency.

[0007] This invention is achieved through the following technical solution:

[0008] A pH-responsive hydrogel microneedle includes a hydrogel microneedle body and colchicine loaded within the hydrogel microneedle body; the hydrogel microneedle body is a three-dimensional network structure hydrogel formed by covalent crosslinking of methacryloyl hyaluronic acid and methacryloyl orthoester.

[0009] The grafting rate of the methacrylamide hyaluronic acid described in this invention is 40%-75%. The higher the grafting rate, the more double bonds can participate in cross-linking, the greater the cross-linking density, and the stronger the mechanical strength and swelling resistance of the hydrogel; however, an excessively high grafting rate will lead to a decrease in the flexibility of the methacrylamide hyaluronic acid, and the microneedles are prone to breakage during puncture.

[0010] Preferably, the mass ratio of the methacrylamide hyaluronic acid to the methacrylamide orthoester is 6:2-8 (for example, it can be 6:2, 6:3, 6:4, 6:5, 6:6, 6:7, 6:8 or any value between the above ratios). This invention controls the mass ratio of methacrylamide hyaluronic acid to methacrylamide orthoester within the above range, resulting in a hydrogel with high mechanical strength prepared by crosslinking.

[0011] The cross-linked methacrylamide hyaluronic acid and methacrylamide orthoester of this invention exhibit excellent swelling properties, enabling more efficient loading of colchicine. Preferably, the colchicine loading in the hydrogel microneedle matrix is ​​0.1-1 mg / 30 mg.

[0012] The pH-responsive hydrogel microneedles of this invention have a regular square pyramidal structure, with a microneedle tip height of 25-1000 micrometers, a microneedle tip diameter of 5-15 micrometers, and a microneedle tip density of 200 tips / cm². 2 -400 roots / cm 2 .

[0013] The present invention also provides a method for preparing the above-mentioned pH-responsive hydrogel microneedles, comprising the following steps:

[0014] S1. Methacrylamide hyaluronic acid, methacrylamide orthoester and photoinitiator Irgacure2959 were dissolved in ultrapure water to obtain a microneedle mixed solution;

[0015] S2. Add the microneedle mixture solution into the microneedle mold, prepare the needle tip and remove air bubbles by vacuum negative pressure, then cure it into a gel by ultraviolet light irradiation, dry and demold to obtain the microneedle body;

[0016] S3. Colchicine solution was added dropwise to the microneedle body, swollen, and dried to obtain pH-responsive hydrogel microneedles.

[0017] The methacrylamide hyaluronic acid described in this invention can be obtained commercially or prepared using conventional methods in the art. For example, it can be obtained by esterification under alkaline conditions (such as adjusting the pH to 8-9 with NaOH solution) using natural hyaluronic acid (HA) as a raw material and methacrylic anhydride (MA) as a modifier.

[0018] In step S1 of this invention, the methacryloyl orthoester is prepared by reacting orthoester monomers (such as diamino orthoester) and methacrylic anhydride as raw materials under the action of a catalyst (such as triethylamine, TEA).

[0019] Preferably, in step S1, the solid content of the methacrylamide hyaluronic acid in the microneedle mixture is 4-6% w / v; the solid content of the methacrylamide orthoester is 2-8% w / v; and the solid content of the photoinitiator Irgacure2959 is 0.1-2% w / v.

[0020] Preferably, in step S2, the wavelength of the ultraviolet light is 230nm-400nm, the irradiation intensity is 100-300w, and the irradiation time is 0.1-10min.

[0021] Preferably, in step S3, the swelling temperature is 0 to -4°C, and the swelling time is 12-24 hours.

[0022] The present invention also provides a hydrogel microneedle patch, comprising the above-described pH-responsive hydrogel microneedles and a backing bonded to the pH-responsive hydrogel microneedles.

[0023] This invention also provides the application of the aforementioned pH-responsive hydrogel microneedle patch in the preparation of a drug formulation for treating acute gouty arthritis. The hydrogel microneedle patch of this invention can be used to treat acute gouty arthritis. The microneedles act on the inflamed site, swelling in the tissue fluid environment, and the hydrogel network structure loosens. Simultaneously, the methacryloyl ester responds to the acidic environment of the inflamed site, gradually eroding the hydrogel microneedle network structure. Colchicine can escape from the hydrogel, and the drug is released into the inflamed site, thereby achieving efficient and rapid anti-inflammatory effects and bone tissue recovery.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] The hydrogel microneedles prepared by this invention using methacrylated hyaluronic acid and methacrylated orthoester as crosslinking components not only possess excellent mechanical properties, swelling capacity, and biocompatibility, but also, relying on the pH sensitivity of methacrylated orthoester, can respond to the pH of the microenvironment at the inflamed site, improving drug utilization. Furthermore, a sustained-release effect can be achieved by altering the degree of crosslinking. This allows for faster and more efficient treatment of acute gouty arthritis, demonstrating significant potential for clinical application. Attached Figure Description

[0026] Figure 1 The image shows the 1H NMR spectrum of HAMA in this invention, with D2O as the deuterated reagent.

[0027] Figure 2 This is a schematic diagram of the hydrogel preparation in Example 1 of the present invention;

[0028] Figure 3 This is a SEM image of the HAMA crosslinked hydrogel and the HAMA-OEMA crosslinked hydrogel in this invention;

[0029] Figure 4 The results show the screening of each component of the hydrogel microneedles in this invention;

[0030] Figure 5 The images shown are of the actual hydrogel microneedles of Example 1 in this invention, along with SEM and LSCM images.

[0031] Figure 6 This is an evaluation of the mechanical strength of the hydrogel microneedles in Example 1 of the present invention;

[0032] Figure 7 This is a test of the skin penetration ability of the hydrogel microneedles in Example 1 of this invention;

[0033] Figure 8 The recovery of skin pores after the hydrogel microneedles of Example 1 of this invention are inserted into the skin;

[0034] Figure 9 Evaluation of the swelling performance of hydrogel microneedles in Example 1 of this invention;

[0035] Figure 10 Images showing the in vitro degradation of hydrogel microneedles in Example 1 and Comparative Example 1 of this invention under different pH conditions;

[0036] Figure 11 This is the standard curve of Col in this invention;

[0037] Figure 12 The drug release of the hydrogel microneedles prepared in this invention under different pH conditions;

[0038] Figure 13The results are for evaluating the cell activity of the hydrogel microparticles prepared in this invention;

[0039] Figure 14 Cell viability / death staining images of the hydrogel microneedles prepared in this invention;

[0040] Figure 15 The results of the blood compatibility evaluation of the hydrogel microparticles prepared in this invention are shown.

[0041] Figure 16 The hydrogel microneedles prepared in this invention are used in vitro;

[0042] Figure 17 This is a schematic diagram of the hydrogel microneedles prepared in this invention used to treat mice with acute gouty arthritis.

[0043] Figure 18 The image shows the changes in knee joint dimensions of mice after treatment with the hydrogel microparticles prepared in this invention, based on the components of the microparticles.

[0044] Figure 19 The hydrogel microparticles prepared in this invention showed the trend of knee joint dimension changes in mice with different components after treatment of inflammatory mice.

[0045] Figure 20 Immunohistochemical analysis of mouse joints was performed after the treatment of acute gouty arthritis in mice with the hydrogel microneedles prepared in this invention was completed.

[0046] Figure 21 The joints of mice were stained with H&E after the treatment of acute gouty arthritis in mice with the hydrogel microneedles prepared in this invention was completed. Detailed Implementation

[0047] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. The described embodiments are only some embodiments of the present invention, and not all embodiments.

[0048] Preparation of methacrylamide hyaluronic acid (HAMA):

[0049] Hyaluronic acid (HA) was dissolved in 50 mL of ultrapure water and stirred continuously overnight. N,N-dimethylformamide (DMF, 50 mL) was added as a co-solvent. 1.19 mL of methacrylic anhydride (MA) was added dropwise, and the pH was maintained between 8 and 9 using 0.5 M NaOH solution. The reaction was carried out at 4 °C for 24 h. Excess acetone was added to precipitate methacryloyl hyaluronic acid (HAMA), and the product was washed three times with ethanol. The product was then redissolved and dialyzed against ultrapure water for 48 h to remove residual organic reagents. Finally, the HAMA product was obtained by freeze-drying, and its repeating unit is:

[0050] Its 1H NMR spectrum is shown below. Figure 1 DS was confirmed to be 75% by NMR.

[0051] Preparation of methacryloyl orthoester (OEMA):

[0052] Under nitrogen protection, 5.59 g (8.10 mol) of diamino orthoester monomer was weighed and added to a 250 mL round-bottom three-necked flask, followed by the addition of an appropriate amount of triethylamine. Then, 8.38 g (54.30 mmol) of methacrylic anhydride was weighed, dissolved and diluted in a small amount of dichloromethane, and added dropwise to the three-necked flask. The reaction was carried out overnight at room temperature. The solvent was removed under reduced pressure. The product was extracted once each with ethyl acetate, 10% K₂CO₃ solution, and saturated NaCl solution. The organic phase was collected, dried, filtered, and removed by rotary evaporation under reduced pressure. The product was then separated by silica gel column chromatography to obtain a yellow oily product, OMA, with the following structural formula:

[0053]

[0054] Example 1: Preparation of pH-responsive hydrogel microneedles

[0055] Preparation process flow as follows Figure 2 As shown, the specific steps include the following:

[0056] S1. Methacrylated hyaluronic acid (HAMA), methacrylated orthoester (OEMA), and photoinitiator Irgacure2959 were dissolved in ultrapure water to obtain a microneedle mixed solution; wherein, the concentration of Irgacure2959 in the microneedle mixed solution was 0.6% w / v; the concentration of HAMA was 6% w / v; and the concentration of OEMA was 8% w / v;

[0057] S2. Add the microneedle mixture solution into the microneedle mold, prepare the needle tip and remove air bubbles by vacuum negative pressure, then cure it into a gel by ultraviolet light irradiation (365nm, 300W, 5min), dry and demold to obtain the microneedle body;

[0058] S3. Add 100 μl of colchicine aqueous solution (10 mg / ml) dropwise to the microneedle body, place it at -4℃ for 12 h to swell, and dry at room temperature to obtain pH-responsive hydrogel microneedles (Col / OE-HMNs).

[0059] Example 2: Screening and optimization of raw materials for each component in hydrogel microneedles:

[0060] The photoinitiator had a solid content of 0.6% w / v, and the HAMA had a solid content of 6% w / v. OMA concentrations of 2%, 5%, 8%, 11%, and 14% w / v were screened, with other conditions the same as in Example 1. After photogelation, the OMA content was screened based on the gelation state and the elastic modulus of the gel measured using a rheometer.

[0061] like Figure 4 As shown, the elastic modulus of the hydrogel increases with increasing OMA content. When the concentration exceeds 8% w / v, OMA undergoes self-crosslinking and cannot form a gel, resulting in a decrease in elastic modulus. Therefore, an OMA concentration of 8% w / v is preferred, as it provides greater mechanical strength to pierce the skin. Based on the above screening, the preferred component contents are: Irgacure2959 0.6% w / v; HAMA 6% w / v; and OMA 8% w / v.

[0062] Comparative Example 1:

[0063] The difference from Example 1 is that methacrylamide orthoester (OEMA) is not added in step S1, while the rest is the same as in Example 1, to obtain hydrogel microneedles (Col / HMNs).

[0064] Comparative Example 2:

[0065] The difference from Example 1 is that methacryloyl ester (OEMA) and colchicine are not added, but otherwise the same as in Example 1, resulting in hydrogel microneedles (HMNs).

[0066] Comparative Example 3:

[0067] The difference from Example 1 is that colchicine was not added, but the rest is the same as in Example 1, resulting in hydrogel microneedles (OE-HMNs).

[0068] Physicochemical characterization of hydrogel microneedles:

[0069] (1) By comparing the SEM images of HAMA after gelation and HAMA-OEMA, see... Figure 3 Compared to cross-linked HAMA, cross-linked HAMA-OEMA has a denser network structure and exhibits different cross-linking morphologies, indicating that HAMA-OEMA has been successfully cross-linked.

[0070] (2) The morphology and structure of the hydrogel microneedles prepared in Example 1 are as follows: Figure 5 As shown, the microneedles were observed using a camera, atomic force microscope, and scanning electron microscope, respectively. The morphology of the microneedles was also observed using a laser confocal microscope after Rhodamine B was dissolved in them. Through these methods, the prepared hydrogel microneedles were found to have a neat array of microneedles with intact tips and a density of 400 microneedles / cm². 2The microneedle tip is a regular square pyramid shape, proving that the microneedle was successfully prepared.

[0071] (3) Test the mechanical strength of the microneedle tip. The test results are as follows: Figure 6 As shown, the mechanical strength of Col / OE-HMNs and Col / HMNs was tested using a universal testing machine. Each microneedle was cut into 5×5 mm pieces, and the samples were placed on a stainless steel plate. A probe moving downwards at a constant speed of 50 mm / s was used to press down 360 micrometers from the needle tip. The needle tip was compressed until it reached 60% of the sample height. During this process, the instrument automatically recorded the force-displacement curve of the probe. The results showed that the maximum pressure that the needle tip of Example 1 could withstand was about 0.5 N, which was sufficient to effectively penetrate the skin. Compared with Comparative Example 1, Example 1 had greater mechanical strength and was easier to penetrate the skin.

[0072] (4) Microneedle penetration ability and skin recovery: After anesthetizing mice with isoflurane, the backs of the mice were shaved. The hydrogel microneedles prepared in Example 1 were then inserted into the skin of the mouse backs and pressed for ten minutes to allow the microneedles to fully penetrate the mouse skin. Figure 7 As shown, after the microneedles were removed, a clear array of needle tips was left on the mouse's back. The recovery of the skin on the mouse's back was then observed. Figure 8 The results showed that the skin on the back of the mice gradually recovered over time, fully recovered at 2 hours, and there was no obvious damage to the skin surface.

[0073] (5) The swelling properties of hydrogel microneedles are shown in [reference needed]. Figure 9 Phosphate-buffered saline (PBS, pH 7.4) was chosen as the swelling medium because it closely resembles the interstitial fluid of the skin, and it has been used in other similar studies to simulate the interstitial fluid. At room temperature, the initial weight of the microneedles was first measured. Subsequently, Col / OE-HMNs were immersed in PBS solution to swell, removed at specific time points, and their surface moisture was blotted with filter paper before weighing. The swelling capacity of the microneedles was calculated according to the Mt-M0 / M0 equation, where M0 and Mt represent the mass of each formulation hydrogel before and after immersion in PBS for 0 to 24 hours. Figure 9 The results show that the hydrogel microneedles prepared in Example 1 can swell to 958% of their own weight in 8 hours, indicating that the hydrogel microneedles have high swelling performance.

[0074] (6) In vitro degradation of hydrogel microneedles: The tips of hydrogel microneedles were immersed in 5 mL of PBS (pH=5.8, pH=7.4) and hyaluronidase (100 u / ml), respectively, and the degradation changes of the tips were observed under an optical microscope at time points of 2, 6, 12, 24, 48, 72 and 96 h.

[0075] Figure 10This describes the in vitro degradation of hydrogel microneedles in Example 1 and Comparative Example 1 of the present invention. From... Figure 10 It can be seen that, under the simulated inflammation environment of pH 5.8, the tip of the microneedle in Example 1 degraded faster than that in Comparative Example 1, and was almost completely degraded after 96 hours. This is because the microneedle in Example 1 contains OMA participating in cross-linking. OMA has acid-sensitive properties; its ester bonds degrade in an acidic environment, thus the microneedle in Example 1 degraded faster, indicating that it is pH-sensitive. At pH 7.4, because OMA is involved in cross-linking in Example 1, its degree of cross-linking is high, and its network structure is denser than that of Comparative Example 1, thus resulting in a slower degradation rate.

[0076] (7) Drug release from hydrogel microneedles:

[0077] Establish a Col standard curve: Dissolve 100 mg Col in 1 mL of PBS buffer to prepare a 100 mg / mL Col solution. Then, dilute the Col solution by half with PBS buffer to obtain Col solutions of different concentrations. Measure the absorbance at 254 nm using an ELISA reader.

[0078] The microneedles of this invention were immersed in 6 mL of PBS and shaken in a shaker for 72 hours. The Col concentration was measured by HPLC.

[0079] The in vitro transdermal absorption characteristics of Col were studied using a diffusion cell. Isolated rat abdominal skin was equilibrated in physiological saline for 30 min and placed on a platform with the dermis facing down and the epidermis facing up. Hydrogel microneedle patches were manually inserted into the skin and fixed between the supply and receiving cells. PBS (pH 5.8) was used as the diffusion medium, automatically filling the receiving cell. The temperature was maintained at approximately 37°C, and magnetic stirring was applied at 600 rpm. At predetermined time points, 1 mL of sample solution was automatically removed from the receiving cell, and 1 mL of PBS was simultaneously injected. The sample was then quantitatively analyzed by HPLC. The cumulative release (Qn) was calculated using the formula Qn / W*100%.

[0080]

[0081] Figure 11 This is the standard curve for Col. Figure 12 This shows the drug release results of the hydrogel microneedles in this invention. From Figure 12It can be seen that, under the simulated inflammation environment of pH 5.8, the drug release rate of the microneedles in Example 1 is faster than that of the microneedles in Comparative Example 1, with complete drug release in 48 hours and a release amount of 85.56%, while the drug release of the microneedles in Comparative Example 1 is complete in 72 hours and a release amount of 79.43%. Furthermore, the drug release of the microneedles in Example 1 differs under the two different pH environments; the drug release rate is faster and the release amount is higher in the acidic environment. This is because the microneedles in Example 1 contain OMA, which can respond to the acidic environment of the inflamed site, causing the hydrogel network structure to be disrupted more quickly, thereby promoting faster drug release.

[0082] (8) Biocompatibility evaluation of hydrogel microneedles:

[0083] Cell compatibility testing: The safety of hydrogel microneedles was assessed using the MTT assay and a double staining assay. Specifically, mouse embryonic fibroblasts (3T3) were seeded into 96-well and 24-well culture plates containing MEM and incubated in a sterile incubator for 12 hours to allow complete cell adhesion. The culture medium in the 96-well plates was slowly removed using a sterile pipette, and then 180 μl of prepared culture medium was added to each well. (200 μl of culture medium was added to the second and third wells to counteract the influence of the culture medium and as a negative control). Then, from the fourth to the ninth well, the extract of the hydrogel microneedles (the hydrogel microneedles were immersed in PBS at pH 5.8 for 48 hours, and the extract was collected) was added as a positive control. Next, the prepared culture plates were incubated in a constant temperature incubator for 24 hours. Finally, the culture medium was removed again, and 200 μl of culture medium was added to the second column of wells. For columns two through nine, 180 μl of the prepared culture medium and 20 μl of MTT solution were added respectively, and incubation continued. After four hours of incubation, the old culture medium was removed with a sterile pipette, and 150 μl of DMSO was added. The plates were then placed on a constant temperature shaker and gently shaken. Finally, the absorbance at 570 nm was measured using a microplate reader. 500 μl of buffer containing 1% Calcein AM and 0.1% pyridine iodide (PI) was added to each well of a 24-well plate. After incubation at room temperature for 30 minutes, the background color was washed away with PBS, and cell viability was observed using an inverted fluorescence microscope. Each group was performed in triplicate.

[0084] Depend on Figure 13 The cytotoxicity assay results of the hydrogel microneedles showed that the cell viability of the hydrogel microneedles in Example 1 and Comparative Example 1 was greater than 80%. Figure 14 Fluorescence images also showed that the cells were predominantly green fluorescent (green: live cells; red blood cells: dead cells), indicating that the hydrogel microneedles prepared in Example 1 and Comparative Example 1 both had good cell compatibility.

[0085] Blood compatibility test: 500 μl of whole blood from mice was collected in an anticoagulant tube, then dispersed in 5 ml of PBS buffer. The mixture was centrifuged at 2500 rpm for 5 minutes, the supernatant was discarded, and the cells were washed three times with PBS. The cells were then resuspended in 10 ml of physiological saline to prepare a red blood cell suspension. Next, 500 μl of the red blood cell suspension was mixed with hydrogel microneedle extract. A negative control group was prepared with 500 μl of red blood cell suspension + 500 μl of physiological saline, and a positive control group was prepared with 500 μl of red blood cell suspension + ultrapure water. Both mixtures were incubated at 37°C and 120 rpm for 1 hour. The supernatant was collected by centrifugation, and the absorbance at 540 nm was measured to calculate the hemolysis rate. Each group was tested in triplicate.

[0086] Figure 15 The hemolysis experiment results of hydrogel microneedles 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, after treatment with hydrogel microneedles prepared in Example 1 and Comparative Example 1, there was no significant difference from the negative control group, and the supernatant was clear. The OD value of each experimental group was detected by a multifunctional enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 540 nm. The hemolysis rate of each group was less than 5%, which proved that no obvious hemolysis occurred and that the microneedles had good blood compatibility.

[0087] (9) Evaluation of the anti-inflammatory properties of hydrogel microneedles:

[0088] In vitro anti-inflammatory performance evaluation of hydrogel microneedles: RAW cells were seeded in 6-well culture plates containing DMEM and cultured in a sterile incubator for 24 h. After discarding the supernatant, the cells were washed twice with PBS, and the medium was replaced with 450 ml of fresh medium containing (1 mg / ml) LPS for 24 h of induction. Simultaneously, 50 μl of different hydrogel microneedle extracts were added for intervention, with 50 μl of PBS as the control group. The cells were incubated in a sterile incubator for 24 h. The culture medium was collected, and the levels of three inflammatory factors were detected using an ELISA kit.

[0089] Evaluation of hydrogel microneedle therapy for acute gouty arthritis: ICR mice were anesthetized with isoflurane (1.5-2% maintenance) for one week, and the knee joint was shaved and fixed. 20 μl of hydrogel microneedle (containing 30 mg / ml MSU) was slowly injected into the joint cavity. Knee swelling occurred, successfully modeling acute gout. Microneedle patches were applied to the right posterior knee joint on day 0 for treatment. The model group served as the control group. The dimensions of the right posterior knee joint of rats in each group were recorded at 0, 6, 12, 24, 48, 72, and 96 hours, and optical images of the hind limb knee joint were recorded at 96 hours. After treatment, HE staining and immunohistochemical analysis of the mouse paws were performed to evaluate the in vivo anti-inflammatory effect of the hydrogel microneedles.

[0090] Figure 16The results show the cytotoxicity evaluation of the hydrogel microneedles prepared in this invention. Compared with the LPS-induced group, both Example 1 and Comparative Example 1 can inhibit the differentiation of RAW cells from M0 to M1 types, while Comparative Examples 2 and 3 have no significant inhibitory effect. Moreover, Example 1 has a higher inhibition efficiency, indicating that the hydrogel microneedles in Example 1 can more effectively reduce inflammation.

[0091] Figure 17 This is a mouse model and treatment diagram of the hydrogel microneedles prepared in this invention for treating acute gout.

[0092] Figure 18 , Figure 19 The results of the experiment on the treatment of acute gout with hydrogel microneedles prepared in this invention are shown. Figure 18 This is a photograph of the knee joint swelling in mice 96 hours after microneedle treatment for acute gout. Example 1 compares with control groups 1, 2, and 3. The knee joint dimension of the group that was closest to the normal group after 96 hours of treatment is shown.

[0093] Figure 19 This shows the trend of knee joint dimension changes in mice during microneedling treatment. From... Figure 18 , Figure 19 It can be seen that the hydrogel microneedles in Example 1 and Comparative Example 1 both have certain anti-inflammatory and swelling-reducing abilities. The hydrogel microneedles in Example 1 can respond to the inflammatory microenvironment of the arthritis site, better promote drug release, and achieve a rapid anti-inflammatory therapeutic effect.

[0094] Figure 20 The immunohistochemical analysis in the mouse joints detected three pro-inflammatory factors: IL-1β, IL-6, and TNF-α. Compared with Comparative Examples 1, 2, and 3, the microneedles in Example 1 showed a more significant effect in inhibiting the pro-inflammatory factors and were closer to the normal group, demonstrating that the pH-responsive hydrogel microneedles prepared in Example 1 have a good anti-inflammatory effect.

[0095] Figure 21 H&E staining was used to observe the histopathological changes of articular cartilage in the knee joint of mice. H&E staining results showed that, compared with the normal group, the articular cartilage surface in the model group was severely damaged, rough, and infected with inflammatory factors. The knee articular cartilage surfaces in Comparative Examples 2 and 3 were also rough, with slight wear, but compared with the model group, the roughness of the articular cartilage surfaces in Comparative Examples 2 and 3 was improved. However, the joint surface in Example 1 was smoother, and its recovery was closer to that of the normal group. This indicates that the hydrogel microparticles prepared in Example 1 have a good therapeutic effect on cartilage damage.

[0096] The above results fully demonstrate that, compared with existing HAMA-based hydrogel microneedles, the HAMA-OEMA cross-linked hydrogel microneedles designed in this invention can penetrate the skin better, have good biocompatibility, and can release drugs in response to the pH environment of the inflamed site, thus achieving the purpose of anti-inflammatory effect more efficiently and quickly.

Claims

1. A pH-responsive hydrogel microneedle, characterized in that, It includes a hydrogel microneedle body and colchicine loaded within the hydrogel microneedle body; the hydrogel microneedle body is a three-dimensional network structure hydrogel formed by covalent crosslinking of methacrylamide hyaluronic acid and methacrylamide orthoester.

2. The pH-responsive hydrogel microneedles according to claim 1, characterized in that, The mass ratio of the methacrylated hyaluronic acid to the methacrylated orthoester is 6:2-8.

3. The pH-responsive hydrogel microneedles according to claim 1, characterized in that, The colchicine loading in the hydrogel microneedle matrix is ​​0.1-1 mg / 30 mg.

4. The method for preparing pH-responsive hydrogel microneedles according to any one of claims 1-3, characterized in that, include The following steps are required: S1. Methacrylamide hyaluronic acid, methacrylamide orthoester, and photoinitiator Irgacure2959 are dissolved in a superconducting solution. A microneedle mixture solution was obtained in pure water; S2. Add the microneedle mixture solution to the microneedle mold, prepare the needle tip using a vacuum negative pressure method, and remove air bubbles. The microneedle body is obtained by photocuring it with ultraviolet light, drying it, and demolding it. S3. Colchicine solution was added dropwise to the microneedle body, swollen, and dried to obtain pH-responsive hydrogel microneedles.

5. The method for preparing pH-responsive hydrogel microneedles according to claim 4, characterized in that, In step S1, the methacryloyl orthoester is prepared by reacting orthoester monomers and methacrylic anhydride under the action of a catalyst.

6. The method for preparing pH-responsive hydrogel microneedles according to claim 4, characterized in that, In step S1, the solid content of the methacrylamide hyaluronic acid in the microneedle mixture is 4-6% w / v; the solid content of the methacrylamide orthoester is 2-8% w / v; and the solid content of the photoinitiator Irgacure2959 is 0.1-2% w / v.

7. The method for preparing pH-responsive hydrogel microneedles according to claim 4, characterized in that, In step S2, the wavelength of the ultraviolet light is 230nm-400nm, the irradiation intensity is 100-300w, and the irradiation time is 0.1-1min.

8. The method for preparing pH-responsive hydrogel microneedles according to claim 4, characterized in that, In step S3, the swelling temperature is 0 to -4℃, and the swelling time is 12-24h.

9. A hydrogel microneedle patch, characterized in that, Includes the pH-responsive hydrogel microneedles as described in any one of claims 1-3 and a backing bonded to the pH-responsive hydrogel microneedles.

10. The use of the pH-responsive hydrogel microneedle patch according to claim 9 in the preparation of a pharmaceutical formulation for treating acute gouty arthritis.

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