Oral hydrogel microspheres targeting the gut for inhibition of gout and methods of making and using the same

Abstract

This invention provides an oral hydrogel microsphere for targeted intestinal inhibition of gout, its preparation method, and its application, belonging to the field of biomedical technology. The preparation method of the oral hydrogel microsphere of this invention includes the following steps: (1) dissolving uricase and dopamine in sodium alginate solution, controlling the concentration of uricase to 10-20 U / mg and the concentration of dopamine to 0.01-0.1 g / ml, and then dispersing by ultrasound; (2) placing the solution obtained in step (1) in a microfluidic device, and using gas microfluidic technology, shearing the solution into uniform droplets under the action of nitrogen flow; (3) dropping the droplets obtained in step (2) into calcium chloride solution, and cross-linking to form the oral hydrogel microsphere for targeted intestinal inhibition of gout. This invention successfully constructs intestinal "bursting" oral hydrogel microspheres loaded with uricase, effectively immobilizing uricase in the intestinal mucosa, thereby inhibiting gout for the first time by excreting free uric acid through the intestine. The microspheres have high uricase activity and good therapeutic effect on gout.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an oral hydrogel microsphere that targets the intestine to inhibit gout, its preparation method, and its application. Background Technology

[0002] Gout is a common inflammatory arthritis caused by hyperuricemia, affecting nearly 4% of adults. Hyperuricemia and secondary gout are primarily influenced by factors related to uric acid metabolism. Uric acid is the final product of purine metabolism in the body, and its levels are maintained in a dynamic balance through the action of the liver, kidneys, and intestines. When this balance is disrupted, serum uric acid levels rise. The inflammatory disease caused by the deposition of sodium urate crystals in and around the joints due to long-term hyperuricemia is called gouty arthritis.

[0003] Uric acid is primarily excreted through the kidneys and intestines. Renal excretion is the main target of current uric acid-lowering drugs. However, high uric acid itself can lead to potential kidney damage, and traditional uric acid metabolism drugs may also exacerbate the burden on the kidneys, resulting in significant limitations in their use. For example, kidney-targeted drugs such as benzbromarone increase the burden on the kidneys and are contraindicated in patients with renal insufficiency; probenecid reduces uric acid reabsorption by inhibiting the activity of renal uric acid transport proteins, but its side effect can cause renal colic. The significant risk of kidney damage, coupled with the fact that drugs targeting the kidneys have been extensively studied and have limited development potential, means that there are currently no effective treatment alternatives for some patients who are not suitable for kidney-targeted drugs.

[0004] The intestines are the second largest site of uric acid excretion after the kidneys, with about one-third of the body's uric acid being excreted through the intestines. Therefore, the intestines have a huge potential for uric acid excretion, effectively relieving pressure on the kidneys. For patients intolerant to renal excretion drugs, intestinal excretion is an important alternative. Furthermore, the process of excreting uric acid through the intestines, along with other bodily waste products, is safer, faster, and more efficient than renal excretion. Therefore, increasing the intestinal uric acid excretion pathway is of practical significance for gout patients.

[0005] However, there are currently no drugs that target the intestinal excretion pathway to lower uric acid and treat gout, so increasing uric acid excretion remains a challenge.

[0006] Similar to the renal excretion pathway, uric acid transporters play a crucial role in intestinal excretion, including the GLUT9 and ABCG2 transporters. Ichida demonstrated, through ABCG2 gene knockout mice, that dysfunctional ABCG2 transporters reduce intestinal urate excretion, a common mechanism in hyperuricemia. DeBosch, through GLUT9 gene knockout mice, demonstrated that mice lacking the intestinal GLUT9 transporter develop hyperuricemic metabolic syndrome. Literature reports that intestinal endothelial cells primarily sense uric acid concentration through TLR receptors. The intestinal cell TLR4-NLRP3 inflammasome and PI3K / Akt signaling pathways regulate the expression of soluble uric acid-stimulated PDZK1 and ABCG2 intestinal uric acid transporters in human intestinal cells. Unlike single-target drugs, the expression of most transporters in these intestinal endothelial cells is related to the uric acid level in the intestinal environment. Studies have found that reducing intestinal uric acid levels has the potential to upregulate the levels of multiple uric acid transporters in intestinal endothelial cells. However, how to efficiently activate the large-scale expression of intestinal uric acid transport proteins is a problem that needs to be solved.

[0007] Meanwhile, uricase, one of the most efficient tools for degrading uric acid, is easily inactivated in the stomach, resulting in low concentrations reaching the intestines. Furthermore, uricase has a short retention time in the intestines and is easily excreted through peristalsis, rendering direct oral administration of uricase ineffective in treating gout. Additionally, lowering the overall intestinal uric acid concentration requires patients to take large doses of uricase, which is challenging for clinical application and limits its widespread use.

[0008] It is evident that, on the one hand, how to efficiently fix uricase to the intestinal wall is a current problem; on the other hand, how to focus on the regulation of uric acid levels in the intestinal mucosal microenvironment, and thereby activate the regulation of overall intestinal uric acid, has great practical significance for the treatment of gout.

[0009] Therefore, how to provide a drug that can effectively target the intestines, efficiently fix uricase to the intestinal wall, fully utilize the drug to regulate the uric acid level of the intestinal mucosal microenvironment, and increase intestinal uric acid excretion to achieve the purpose of treating gout has become an urgent technical problem to be solved. Summary of the Invention

[0010] This invention aims to solve the aforementioned technical problems by providing an oral hydrogel microsphere for targeted intestinal inhibition of gout, its preparation method, and its application. The technical objective of this invention is to provide, for the first time, a hydrogel microsphere and its drug delivery system that inhibits gout by excreting free uric acid through the intestines, achieving highly efficient utilization of uricase activity, fully leveraging its role in regulating uric acid levels in the intestinal mucosal microenvironment, significantly increasing intestinal uric acid excretion, and thus substantially improving the therapeutic effect on gout.

[0011] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0012] One objective of this invention is to provide a method for preparing oral hydrogel microspheres that target the intestines to inhibit gout, comprising the following steps:

[0013] (1) Dissolve uricase and dopamine in sodium alginate solution, control the concentration of uricase to be 10-20 U / mg and the concentration of dopamine to be 0.01-0.1 g / ml, and disperse by ultrasonication;

[0014] (2) Place the solution obtained in step (1) in a microfluidic device and use gas microfluidic technology to shear the solution into uniform droplets under the action of nitrogen flow.

[0015] (3) The droplets obtained in step (2) are dropped into a calcium chloride solution and cross-linked to form the oral hydrogel microspheres that target the intestine to inhibit gout.

[0016] The key to the preparation method of the present invention lies in the use of uricase and dopamine in combination, and by controlling the concentration of uricase and dopamine, the activity of uricase is well guaranteed. This fully utilizes the regulation of uric acid excretion by uricase in the intestine, and reduces the dosage and frequency of administration of uricase, thus significantly improving the therapeutic effect on gout.

[0017] The inventors' exploratory experiments showed that when only dopamine was present in the aforementioned hydrogel microsphere system, it was impossible to regulate intestinal uric acid excretion; when only uricase was present in the aforementioned hydrogel microsphere system, the activity of uricase was low, failing to fully exert its effect in regulating intestinal uric acid excretion, leading to increased uricase dosage, more frequent administration, and poor efficacy. However, when both uricase and dopamine were present in the aforementioned hydrogel microsphere system, and combined according to the concentration of the present invention, the activity of uricase could be maximized, significantly improving its effect in regulating intestinal uric acid excretion. The activity of uricase was nearly three times higher than when only uricase was present, thereby reducing the dosage and frequency of uricase administration and significantly improving efficacy.

[0018] The inventors have successfully constructed oral hydrogel microspheres loaded with uricase for intestinal "bursting" using the above method, effectively immobilizing uricase in the intestinal mucosa and thus, for the first time, inhibiting gout by excreting free uric acid through the intestine. After oral administration, the uricase released from the hydrogel microspheres hydrolyzes uric acid to produce hydrogen peroxide. This hydrogen peroxide reacts with catalase in the intestine to create a local oxygen microenvironment, inducing dopamine polymerization into polydopamine, thereby immobilizing further released uricase on the small intestinal wall. Through this cascade reaction, a small amount of uricase can upregulate the expression of uric acid transport proteins in intestinal endothelial cells, expelling uric acid from the endothelial cells into the intestine and excreting it through feces, thus reducing the body's uric acid levels.

[0019] In vitro experiments and in vivo studies in mice revealed that the intestinal "bursting" hydrogel microspheres enabled precise intestinal delivery of dopamine and uricase. In vitro experiments using fecal samples from clinical gout patients and pig small intestines demonstrated that the microspheres significantly reduced fecal uric acid levels by up to 37%. Experiments in mouse models of hyperuricemia and acute gouty arthritis confirmed that the microspheres effectively targeted intestinal uric acid excretion in hyperuricemic mice and arthritic joints in mice with gouty arthritis, effectively increasing fecal uric acid excretion by 27.6% and reducing serum uric acid by 71%. Furthermore, 16S ribosomal RNA sequencing showed that the microspheres optimized the gut microbiota composition, significantly increasing probiotics such as *Muciniphila* and *Verrucomicrobiales*, while inhibiting common harmful bacteria in the gut of gout patients, such as *Bacteroidetes*, thereby improving intestinal homeostasis. In summary, we have achieved, for the first time, a treatment for gout through uric acid excretion via the small intestinal mucosa.

[0020] Furthermore, in step (1), the concentration of the uricase is controlled to be 10 U / mg.

[0021] Furthermore, in step (1), the concentration of dopamine is controlled to be 0.01 g / ml.

[0022] Furthermore, the concentration of sodium alginate in step (1) is 1% w / v.

[0023] Furthermore, in step (2), the flow rate of nitrogen is controlled to be 0.2 to 1.0 L / min, preferably 0.8 L / min.

[0024] Furthermore, in step (2), the flow rate of the solution obtained in step (1) is controlled to be 10-30 mL / h, preferably 20-25 mL / h.

[0025] Furthermore, the concentration of the calcium chloride solution in step (3) is 100 mM.

[0026] The second objective of this invention is to provide an oral hydrogel microsphere that targets the intestines to inhibit gout, prepared by the method described above.

[0027] As a preferred embodiment, the diameter of the hydrogel microspheres is 180-220 micrometers.

[0028] A third objective of this invention is to provide the application of the above-mentioned oral hydrogel microspheres that target the intestine to inhibit gout in the preparation of drugs for treating gout.

[0029] The beneficial effects of this invention are as follows:

[0030] (1) This invention is the first to achieve the inhibition of gout by excreting free uric acid through the intestine, and the oral hydrogel microspheres loaded with uricase loaded with uricase effectively immobilize uricase in the intestinal mucosa and efficiently exert the activity of uricase.

[0031] (2) The oral hydrogel microspheres of the present invention can target the intestine well and can efficiently fix uricase to the intestinal wall, giving full play to the role of uricase in regulating the uric acid level of the intestinal mucosal microenvironment. The combination of uricase and dopamine better ensures the activity of uricase, thus achieving the effect of highly effective treatment of gout. Attached Figure Description

[0032] Figure 1 This is a schematic diagram of the synthesis process of UPA microspheres.

[0033] Figure 2 A schematic diagram illustrating the use of in-situ intestinal mucosal regulation to upregulate uric acid excretion and radically treat gout: A) A pH-responsive micro / nano hydrogel microsphere system (UPA) encapsulating uricase / PDA in a calcium alginate shell was developed using gas microfluidics and ion crosslinking technology; B) Oral administration of UPA microspheres achieved gastric protection and sustained release in the small intestine, realizing in-situ dopamine polymerization in the small intestinal mucosa, thereby achieving in-situ immobilization of uricase in the small intestine; C) Through this in-situ immobilization of uricase in the mucosa, in-situ uric acid regulation was achieved, significantly upregulating uric acid transport proteins in endothelial cells; D) This further activated intestinal excretion of uric acid and improved gout symptoms.

[0034] Figure 3 Three different sizes of microspheres were prepared for this example, with a scale bar of 100 μm.

[0035] Figure 4The images are: A) Scanning electron microscope images of UPA microspheres of different sizes, with scale bars of 500 μm, 50 μm, and 10 μm from left to right; B) Particle size distribution of UPA microspheres; C) CLSM images of the shell-core structure of Alg / Uricase microspheres, Rho B (red) modified alginate (shell) and FITC (green) modified uricase (core), scale bar 50 μm; D) Microsphere diameter variation at different nitrogen flow rates; E) FTIR results; F) Representative images of the degradation process of UPA microspheres in AGF, ASF, and ACF, scale bar 100 μm; G) Schematic diagram of the cascade reaction; H) Size change curves of UPA microspheres in AGF, ASF, and ACF; I) Release curves of uricase from UPA microspheres in AGF, ASF, and ACF, respectively; J) Swelling rate of UPA microspheres in AGF, ASF, and ACF (n=3).

[0036] Figure 5 This study investigates the degradation of UPA microspheres in AGF, ASF, and ACF solutions in vitro.

[0037] Figure 6 The following are examples: A) Schematic diagram of UPA microsphere adhesion experiment in isolated porcine intestine; B) Reaction of porcine ileum in UPA microsphere solution, with polydopamine adhering to the surface of the porcine intestine after 3 hours; C) Effect of different microsphere sizes on the reduction of uric acid content in isolated porcine intestine experiment; D) Effect of different dopamine contents on the reduction of uric acid content in isolated porcine intestine experiment; E) Effect of different uricase contents on the reduction of uric acid content in isolated porcine intestine experiment; F) The reduction in uric acid content in feces of gout patients; G) The rate of uric acid reduction in feces of gout patients; H) Schematic diagram of in vitro reaction of feces from clinical gout patients with UPA microsphere solution, followed by centrifugation and detection of uric acid content in the supernatant after 24 hours.

[0038] Figure 7 The antibacterial activity of different microspheres was determined using the plate coating method.

[0039] Figure 8 The objectives were as follows: A) and B) Flow cytometry analysis of RAW 264.7 cells containing M1 and M2 macrophages, incubated with uric acid, UA, and UPA to determine the in vitro anti-inflammatory properties of UPA; C) Quantitative RT-PCR detection of mRNA expression of genes related to ABCG2 intestinal uric acid transporter, GLUT9 intestinal uric acid transporter, caspase-1, PI3K, TLR4, TLR2, TNF-α, and IL-1β in Caco-2 cells (n=3); D) Representative Western blot results of Caco-2 cell-related genes ABCG2 and GLUT9 intestinal uric acid transporter; E) Quantitative analysis of protein levels obtained from Western blot analysis (n=3).

[0040] Figure 9 For: A) Immunofluorescence double staining of F4 / 80 (green) and CD86 (red), scale bar, 25 μm; B) Immunofluorescence double staining of F4 / 80 (green) and CD206 (red), scale bar, 25 μm; C) and D) Semi-quantitative analysis of the optical density of CD86 and CD206 (mean ± SD, ***P<0.001 and ****P<0.0001; n=5); E) and F) Levels of inflammatory factors TNF-α and IL-1β in RAW 264.7 cells treated with uric acid, UA and UPA.

[0041] Figure 10 For: A) and B) Caco-2 and RAW 264.7 cells treated with uricase, UA, and UPA, calcein-AM / PI double staining; C) ROS levels in RAW 264.7 cells with or without UA; D) ROS levels in RAW 264.7 cells, with UPA and uricase concentrations of 100 ppm and 20 ppm, respectively. Scale bar, 400 μm.

[0042] Figure 11 To determine the toxicity of different concentrations of uricase, UA, and UPA extracts at 24h and 48h using the CCK-8 assay (n=4).

[0043] Figure 12 The objectives were as follows: A) Experimental design: Mice were administered hypoxanthine solution by gavage for 7 consecutive days. One hour after hypoxanthine administration, potassium oxychloride was injected intraperitoneally. Each mouse was given 4 U / day (200 U / kg) of uricase orally. The uricase content was the same in all experimental groups. B) Intestinal tissue PCR. C) Changes in H2O2 concentration in mouse intestines. D) Changes in urinary uric acid concentration in mice. E) Changes in serum uric acid concentration in mice. F) Changes in fecal uric acid concentration in mice. G) Changes in water intake in the Sham, HUA, Uricase, UA, and UPA groups. H) Detailed recording and analysis of daily weight changes (mean ± SD, ****P < 0.0001, n = 7).

[0044] Figure 13The study included: A) Representative Micro-CT images of the ankle joints in the Sham, MSU, Uricase, UA, and UPA groups; B) Gait analysis of mice with MSU-induced acute gout induced by UPA, where mice with red ink forelegs and blue ink for hindlegs had to run across a 100 cm long and 10 cm wide track covered with paper, with the dashed line representing stride length; C) Quantification of stride length at 6 and 24 hours, expressed as mean ± SD (n = 7); D) Intestinal fluorescence images at different time points after oral administration of FITC-Uricase or FITC-UPA; Statistical differences were analyzed by one-way ANOVA and t-test: *p < 0.05, **p < 0.01, and ns (not significant).

[0045] Figure 14 To enhance the expression of two intestinal transport proteins, ABCG2 and GLUT9, in UPA, the following images were used: A)-B) Representative hematoxylin and eosin (H&E) staining images of intestinal tissues and major organs in each group, scale bar: 100 μm, n=7; C) Representative immunohistochemical staining of ABCG2, scale bar: 100 μm; D) Representative immunohistochemical staining of ABCG2 and GLUT9, scale bar: 100 μm; E) Intestinal injury score of each group based on H&E staining analysis, n=7; F) Semi-quantitative analysis of ABCG2 immunohistochemical staining; G) Representative images of Western blot analysis of ABCG2 and GLUT9 in mouse small intestinal tissue; H) Quantification of relative ABCG2 and GLUT9 levels using ImageJ software; I) Representative images of Western blot analysis of the upper, middle, and lower ileum segments of mice in the Sham, HUA, and UPA groups; J) Quantification of relative ABCG2 and GLUT9 levels in the upper, middle, and lower ileum segments of mice in the three groups using ImageJ software.

[0046] Figure 15 To assess biocompatibility in vivo, H&E staining of heart, lung, liver, and kidney tissues from each group was performed to demonstrate the biocompatibility of UPA in mice. Scale bar: 500 μm.

[0047] Figure 16 The objectives are as follows: A) Experimental design: Synthetic MSU was injected intra-articularly into the right ankle joint of Balb / c mice to establish an acute gout model; B) Changes in ankle joint diameter in each group of mice (mean±SD, ****P<0.0001, n=7); C) Representative photographs of the hind paws of different treatment groups at 6h and 24h after ankle joint injection; D) H&E staining images of the synovial region in different groups of mice with MSU-induced acute gout, with scale bars of 50μm and 200μm for the top and bottom images, respectively.

[0048] Figure 17 The effect of different flow rates of sodium alginate solution on the size of hydrogel microspheres. Detailed Implementation

[0049] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is described in detail below with reference to embodiments. It should be noted that the following embodiments are for explanation and illustration only and are not intended to limit the invention. Non-essential improvements and adjustments made by those skilled in the art based on the above description are still within the scope of protection of this invention.

[0050] Example 1

[0051] (I) Experimental Methods

[0052] 1. Synthesis of hydrogel microspheres

[0053] Hydrogel microspheres were prepared using gas microfluidics technology, with the sodium alginate (Alg) solution concentration adjusted to 1% w / v and the nitrogen flow rate set at 0.8 L·min. -1 To ensure the microspheres have good morphology and uniform particle size, the flow rate of sodium alginate is controlled at 10-30 mL / h. -1 The coaxial needle is perpendicular to the ground, with a receiving distance of at least 9 cm. Sodium alginate solution is injected using the coaxial needle, and then sheared into uniform droplets under a nitrogen gas flow. The droplets are then dropped into a 100 mM calcium chloride (CaCl2) solution by gravity. Alg is reacted with Ca... 2+ Cross-linking leads to the formation of the core of the hydrogel microspheres. The Alg hydrogel microspheres were collected, and any remaining ions on the surface were washed twice with ddH2O and stored at 4°C in the dark.

[0054] Uricase was dissolved in sodium alginate (Alg) solution and sonicated for 30 minutes to form a uniformly dispersed uricase and Alg solution. The remaining steps were performed as described above to prepare calcium alginate / uricase composite hydrogel microspheres (denoted as UA).

[0055] Uricase and dopamine were dissolved in sodium alginate (Alg) solution and sonicated for 30 minutes to form a uniformly dispersed uricase and PDA / Alg compound. The remaining steps were performed as described above to prepare calcium alginate / uricase / dopamine composite hydrogel microspheres (denoted as UPA).

[0056] 2. Characterization of microspheres

[0057] The material structure was verified using a Fourier transform infrared spectroscopy (Nicolet 6700). The microspheres were freeze-dried and observed using a scanning electron microscope (SEM). The morphology of the microspheres was observed using an optical microscope (LSM800, Zeiss, Germany), and the diameter of the microspheres was measured using Image J. The microspheres were observed under a confocal microscope using two fluorescent methods: one using Rhodamine B (Rho B) to label sodium alginate, and the other using FITC-labeled uricase.

[0058] 3. In vitro human fecal experiment

[0059] Human tissue specimens were obtained from 12 donors of different ages and sexes. Microsphere diameter testing: UPA of different diameters were prepared using a nitrogen gas flow method, with diameters of 100 μm, 200 μm, 400 μm, 800 μm, 1 mm, and 2 mm. The microspheres were placed in an environment containing uric acid, artificial small intestinal fluid, and human feces to study the residual uric acid in the feces.

[0060] 4. Isolated porcine small intestine experiment

[0061] Pig tissues were obtained from the slaughterhouse. Pigs were euthanized, and fresh tissues were removed and stored on ice. Tissue (4 cm²) was exposed to uric acid and microsphere solution (10 mL), and washed three times with PBS buffer (1X) to remove excess PDA. Microspheres were placed in environments containing uric acid, artificial small intestinal fluid, and isolated pig small intestine, and the residual uric acid in the pig small intestine was studied at different pH solutions and time points.

[0062] 5. Swelling and Degradation Experiments

[0063] The swelling behavior of microspheres was studied by measuring their water absorption capacity using the immersion method. 10 mg of lyophilized microspheres were placed in a 5 mL centrifuge tube and weighed (M1). Then, 2 mL of AGF (artificial gastric juice), ASF (artificial small intestinal juice), and ACF (artificial colonic juice) were added, and the mixture was continuously shaken (100 rpm, 37°C). At the corresponding time points, after centrifugation, the supernatant was removed, excess liquid was wiped off with filter paper, and the microspheres were weighed (Mt). M0 represents the initial mass of the microspheres. The degree of swelling of the microspheres was calculated using the following formula:

[0064] Swelling degree(%)=[(Mt-M1) / M0]*100

[0065] Microspheres were immersed in simulated gastric fluid (2 hours), simulated small intestinal fluid (4 hours), and simulated colonic fluid, respectively. Diameter was monitored at different time points. To investigate the degradation process of the microspheres, the degradation was observed under an optical microscope at different time points (30 minutes, 1 hour, 2 hours, 6 hours, 24 hours, and 36 hours). Microspheres were placed in simulated gastric and intestinal fluids to assess their degradability. Microspheres were suspended in 2 mL of AGF, ASF, and ACF and placed on a shaker (100 rpm, 37°C). Morphological changes of the microspheres were observed under an optical microscope at the corresponding time points.

[0066] 6. In vitro release study

[0067] 100 mg of microspheres were immersed in a solution containing 10 mL of AGF (containing 0.1% Tween 80) (M W The microspheres were placed in dialysis bags containing 3500 Da, then immersed in 40 mL of the same release buffer and shaken continuously (100 rpm, 37°C). Then, 2 mL of external release buffer was collected every 0.5 hours, with the same volume of buffer added. After 2 hours, the microspheres were sequentially transferred to ASF (containing 0.1% Tween 80) and ACF (containing 0.1% Tween 80), with buffer collected every hour. The concentration of uricase in each sample was measured using a UV spectrophotometer (Eppendorf, Germany), and release curves were plotted.

[0068] The release behavior of uricase in AGF, ASF, and ACF was also examined. The concentration of uricase in each sample was measured sequentially at 1, 2, 4, 6, 8, 10, 12, and 24 hours, and then a 24-hour release curve was plotted.

[0069] 7. Antibacterial test

[0070] Plate count method: *Escherichia coli* and *Staphylococcus aureus* were grown in Luria-Bertani (LB) medium, respectively. Different groups were treated with calcium alginate microsphere extract, calcium alginate uricase microsphere extract, and dopamine-uricase-calcium alginate extract, respectively. All cultures were incubated at 37°C in a shaking incubator for 24 hours, then diluted on diffusion plates (LB medium). After incubation at 37°C for 12 hours, colonies on the plates were counted to analyze the antibacterial activity of the microspheres. The experiment was repeated at least three times.

[0071] 8. Biocompatibility evaluation

[0072] Cell counting kit-8 (CCK-8) and calcein acetoxymethyl ester / propidium iodide (calcein AM / PI) cell viability / cytotoxicity assay kit were used to test the biocompatibility of calcium alginate microspheres. Caco-2 cells and RAW264.7 cells were seeded in 24-well plates (1 × 10⁶ cells per well). 4Caco-2 cells and RAW264.7 cells were seeded in 96-well plates (1 × 10⁶ cells per well) and co-cultured with various microsphere extracts at 37°C and 5% CO₂. On days 1, 2, and 3, cells were incubated with Calcein AM / PI buffer (Proteintech Co., Ltd., China) for 15 minutes, followed by observation under a fluorescence microscope (Nikon ECLIPSE Ts2R, Japan). 4 Cells were cultured at 37°C and 5% CO2 with various microsphere extracts. After 1, 2 and 3 days of culture, the cells were incubated for 1 hour in a medium containing 10% CCK-8 (Dojindo, Japan), and then the absorbance at 450 nm was measured by enzyme labeling (Molecular Devices, Japan).

[0073] 9. ROS testing

[0074] Further investigation was conducted on the inflammatory stress in RAW264.7 cells during treatment with uricase, uricase microspheres, and uricase-dopamine microspheres for hyperuricemia. Intracellular reactive oxygen species (ROS) levels in RAW 264.7 cells were monitored using 2',7'-dichlorofluorescein diacetate (DCFH-DA), a non-fluorescent reagent (DCF) that reacts with intracellular ROS and produces fluorescent 2,7-dichlorofluorescein. The fluorescence intensity of DCF was correlated with the intracellular ROS content. Upon addition of uric acid, uricase-treated cells exhibited significant green fluorescence, indicating substantial ROS production. Simultaneously, RAW cells treated with uricase-dopamine microspheres showed negligible green fluorescence, suggesting that the use of uricase-dopamine microspheres significantly reduced the impact of ROS. These results strongly suggest that the designed uricase-dopamine microspheres have minimal toxicity in the treatment of hyperuricemia.

[0075] 10. Macrophage polarization analysis

[0076] RAW 264.7 cells (3 × 10⁶ cells per well) 5Cells were seeded separately in confocal culture dishes and incubated with microspheres-FITC or PBS (control) at 37°C at different time points. After washing three times with cold PBS, the cells were digested with trypsin and then resuspended in 200 μL PBS for analysis using a flow cytometry system (BD FACSVerse). For morphological observation, cells in the confocal culture dishes were fixed with 4% PFA and stained with F-actin (Solarbio, China) and observed under a confocal laser microscope (UltraVIEW VoX, Perkin-Elmer, USA). FITC Anti-nouse CD86 (biolegend), Integrin aM / CD11b antibody (OX42) (Santa Cruz Biotechnology), CD206 / Mannose Receptor / MMR antibody (15-2) (Santa Cruz Biotechnology), CD86 (Santa Cruz Biotechnology), CD206 (Santa Cruz Biotechnology), F4 / 80 (CellSignaling), Alexa Fluor 488-labeled goat anti-mouse IgG (H+L) (Beyotime), Alexa Fluor 647-labeled goat anti-rabbit IgG (H+L) (Beyotime).

[0077] 11. Real-time quantitative PCR

[0078] RAW 264.7 and Caco-2 cells were seeded in 6-well plates (1 × 10⁶ cells per well). 5 Cells were collected and treated with a medium containing uric acid and uricase. Total RNA was extracted and cDNA was obtained using TRIzol reagent (Invitrogen Co., Ltd., Grand Island, NY) and PrimeScript Reverse Transcriptase Kit (Takara Bio Inc., Japan), respectively, according to the manufacturer's instructions. Real-time PCR was performed using SYBR Premix Ex Taq (Takara Bio Inc., Japan).

[0079] Primer sequences are shown in Table 1 below:

[0080] Table 1

[0081]

[0082] 12. Western blot analysis

[0083] Caco-2 was inoculated into 6-well plates (1 × 10⁻⁶ per well). 5 Cells were lysed in a medium containing uric acid and uricase. Cells were lysed on ice for 0.5 hours using RIPA lysis buffer (Servicebio Co., Ltd., China) containing protease inhibitors, and the supernatant was collected by centrifugation. The supernatant was separated by SDS-PAGE gel electrophoresis, transferred to a PVDF membrane (Millipore, Billerica, MA), blocked with 5% BSA (Servicebio Co., Ltd., China) at room temperature for 1 hour, and incubated overnight at 4°C with primary antibodies including anti-ABCG2 (Protein Technology Inc.) and anti-SLC2A9 (Protein Technology Inc.). After incubation with the corresponding secondary antibodies (Servicebio Co., Ltd., China) for 1 hour, the blot was detected by a chemiluminescence system, and the optical density was measured using ImageJ software.

[0084] 13. Establishment of a hyperuricemia model and drug management

[0085] Animal experiments were approved by the Animal Research Committee of Shanghai Jiao Tong University School of Medicine (SYXK 2018-0027). The animal model was established in Balb / c mice. Mice were first gavaged with 500 mg / kg hypoxanthine solution for 7 consecutive days. One hour after hypoxanthine administration, potassium oxychloride (250 mg / kg) was injected intraperitoneally. Hypoxanthine and potassium oxychloride were suspended in 0.5% sodium carboxymethyl cellulose (CMC-Na). The control group was orally administered the same amount of 0.5% CMC-Na. Each mouse was orally administered 4 u / day (200 U / kg) of uricase. The uricase content of the experimental groups was the same. The experiment was divided into the following five groups (n=10 in each group): (1) normal group as negative control group, (2) hyperuricemia mouse model group as positive control group without treatment, (3) hyperuricemia mouse model group + uricase group, (4) hyperuricemia mouse model + UA microspheres, (5) hyperuricemia mouse model + UPA microspheres. At specific time intervals, changes in water intake, food intake, and body weight of mice were monitored, and blood samples (50 μl) were collected from the orbital rim and serum UA concentration was measured using a uric acid assay kit.

[0086] 14. Biochemical analysis of serum, feces, and urine; determination of intestinal H2O2 production.

[0087] Blood samples were collected from the heart on day 7. The collected samples were immediately transferred to ice and centrifuged at 4°C for 15 minutes after 1 hour. Serum was separated and stored at -80°C until analysis. Uric acid, creatinine, and blood urea nitrogen (BUN) levels, as well as inflammatory markers in the blood, such as changes in monocyte / macrophage, neutrophil, and white blood cell counts, were measured using a biochemical hematology analyzer. Fecal samples were collected on days 2, 4, and 7 and dried at 60°C. Approximately 0.100 g of feces was added to 4 volumes of PBS solution, sonicated for 10 min, and incubated at 100°C for 30 min and 37°C for 120 min, respectively. The homogenate was centrifuged at 10000 rpm for 10 min, and the supernatant was separated. Fecal uric acid (Fua) levels were measured using a uric acid kit. All procedures were performed according to the kit instructions. The intestines were dissected immediately after euthanasia, and feces were collected in tubes and stored at -80°C before use. H2O2 concentration was determined during the degradation of UA in vivo. Small intestinal fluid was collected to measure H2O2 levels. After collection, H2O2 levels were detected using an H2O2 assay kit according to the manufacturer's instructions.

[0088] 15. H&E staining, immunohistochemistry

[0089] Tissue samples were fixed in 4% paraformaldehyde. After dehydration in ethanol, the tissues were embedded in paraffin and sectioned to a thickness of 4 μm. Histopathological features were assessed by H&E staining. Tissue damage scores were evaluated based on previous reports. For immunohistochemistry, tissue sections were deparaffinized, rehydrated, and rinsed, followed by antigen retrieval (thermally induced epitope retrieval) and blocking (goat serum). Next, tissue sections were incubated overnight with primary antibodies, followed by biotinylated secondary antibodies (for immunohistochemical staining). The following primary antibodies were used: anti-ABCG2 (Protein Technology Inc.) and anti-SLC2A9 (Protein Technology Inc.). Immunohistochemical images were scored using ImageJ software.

[0090] 16. RNA extraction, quantitative real-time PCR (qRT-PCR), and Western blotting.

[0091] Intestinal tissue samples were collected on day 7. Collected samples were immediately transferred to liquid nitrogen and stored at -80°C until analysis. The tissues were homogenized in liquid nitrogen, and protein concentration was determined using the BCA protein quantification method. Protein loading buffer was used to quantify the protein to 5 mg / ml, and the samples were stored at -20°C for later use. Repeated freeze-thaw cycles were avoided. The supernatant was separated by SDS-PAGE gel electrophoresis, transferred to a PVDF membrane (Millipore, Billerica, MA), blocked with 5% BSA (Servicebio Co., Ltd., China) at room temperature for 1 hour, and incubated overnight at 4°C with primary antibodies including anti-ABCG2 (Protein Technology Inc.) and anti-SLC2A9 (Protein Technology Inc.). After incubation with secondary antibodies for 1 hour, the blot was detected using a chemiluminescence system, and the optical density was measured using ImageJ software.

[0092] An appropriate amount of the sample to be tested was added to liquid nitrogen and then ground. Total RNA extraction and cDNA acquisition were performed using TRIzol reagent (Invitrogen Co., Ltd., Grand Island, NY) and PrimeScript Reverse Transcriptase Kit (Takara Bio Inc., Japan) according to the manufacturer's instructions, respectively. Real-time PCR was performed using SYBR PremixEx Taq (Takara Bio Inc., Japan).

[0093] 17. Gut microbiota 16S rRNA sequencing analysis

[0094] To investigate whether and how UPA affects the composition or abundance of the gut microbiota, we performed an in-depth analysis of the bacterial composition of the mouse gut using 16S rRNA sequencing. Fecal samples were collected from each mouse on day 7 and stored at -20°C. Total DNA was extracted using the EZNA Soil DNA Kit (OmegaBio-Tek, Norcross, GA, USA). Microbiota composition was determined on the Illumina MiSeq platform (Illumina, San Diego, USA) following the protocols of Majorbio Bio-Pharm Technology Co., Ltd. Operational taxonomic units (OUT) were clustered in UPARSE (version 7.1). The classification of OUT representative sequences was then evaluated using the RDP classifier. All data were ultimately analyzed on the Majorbio Biocloud platform.

[0095] 18. IVIS and Tissue Distribution

[0096] To facilitate observation of drug distribution in vivo, improvements were made to the intestinal targeted delivery system (microspheres) and the non-targeted delivery system (uricase) with FITC. Eight-week-old female Balb / c mice were selected as animal models for tissue imaging. Briefly, mice were fasted overnight and then force-fed with either FITC-uricase or FITC-UPA. At 1 and 4 hours, the intestines of the mice were collected after anesthesia and observed using IVIS (PerkinElmer, Inc., Shelton, CT).

[0097] 19. Establishment and drug management of an acute gouty arthritis model

[0098] Preparation of MSU: 1 g UA was dissolved in 200 mL of water containing 6 mL of 1 M NaOH at 70 °C. The pH of the resulting solution was adjusted to 7.1-7.2 with HCl or NaOH. The final solution was allowed to cool naturally and stirred slowly at room temperature, then stored overnight at 4 °C. The precipitate was filtered from the solution, dried at low temperature, and sieved through a 250 μm metal mesh. MSU-induced acute gout model: Under isoflurane anesthesia, MSU (20 mg / mL, 50 μL) was injected intra-articularly into the right ankle joint of mice to induce an acute gout model. The normal group was injected with normal saline (100 μL). Then, MSU (50 μL) was injected intra-articularly into the joint cavities of the MSU group, MSU+Uricase, MSU+UA, and MSU+UPA groups, respectively. On day 5, the circumference diameter of the right ankle joint of each mouse was measured twice using the joint circumference method, and the average value was taken as the normal pre-inflammatory joint circumference for each mouse. Mice were anesthetized with 45 mg / kg 2% sodium pentobarbital. The mice were then fixed supine on an operating table, and the hair on the right ankle joint was removed under aseptic conditions. The ankle joint was gently flexed, and a needle was inserted through the middle of the joint to inject 0.2 ml of sodium urate solution (2.5 g / 100 ml) into the right ankle joint of the model mouse to establish the AGA model. The mouse was given the drug at a standard dose of 100 g / ml, once daily for 7 days. The ankle joint model was established on day 5 after gavage, and the drug was continued for 2 days after modeling. The synovial tissue of the ankle joint was harvested on day 7. The diameter of the ankle joint was monitored using calipers to assess the distance between the medial and lateral condyles in each group.

[0099] 20. Gait Analysis

[0100] Gait analysis was performed to assess behavior in the affected limb due to acute gout. Each mouse was placed on a 100 cm × 10 cm track and allowed to move freely from one side to the other without any additional stimulation. The forepaws of each mouse were stained red and the hind paws blue to record their footprints.

[0101] 21. Microscopic CT scan

[0102] Micro-CT analysis was performed on ankle joint samples to evaluate the uric acid-lowering effect of the microspheres in vivo. Ankle joints were fixed in 4% PFA and placed on a Micro-CT (Skyscan Co., Belgium) sample examination plate for scanning and 3D reconstruction (resolution: 9 μm, voltage: 50 kV, source current: 500 mA, rotation step length: 0.5°). Regions of interest (ROIs) were selected in the cortical bone of the mid-shaft of the ankle joint and the cancellous bone of the distal femur.

[0103] 22. Histological analysis

[0104] Mouse ankle joints were fixed in 10% formalin for 7 days, decalcified in 15% EDTA-buffered saline for 2 months, dehydrated, embedded in paraffin tissue, and cut into 3-micrometer-thick sections. The sections were stained with Hematoxylin and eosin (H&E) to observe the inflammatory infiltration of the synovium. Images were obtained under an optical microscope.

[0105] 23. Statistical Analysis

[0106] Statistical data were processed and analyzed using GraphPadPrism 7.0 (GraphPad, USA) and SPSS software version 19.0 (IBM, USA). Quantitative data were expressed as mean ± standard deviation or median, and categorical data were expressed as numbers (percentages). Analysis of variance and the Mann-Whitney U test were used to compare differences between two or more groups. Finally, p < 0.05 was considered statistically significant.

[0107] (II) Experimental Results

[0108] 1. Characterization of microspheres

[0109] First, a calcium alginate / uricase / dopamine composite microsphere (UPA) with intestinal-targeted blasting properties was prepared. Figure 1 By accelerating dopamine polymerization through a "burst" response in the small intestinal environment, in-situ fixation of uricase in the small intestinal mucosa was achieved, thereby enabling intervention in uric acid levels within the small intestinal mucosal microenvironment. Figure 2 Hydrogel microspheres were prepared using gas microfluidics. Dopamine, carrying a positive charge, was bound within a calcium alginate shell. The microspheres were then ionically cross-linked to cover the calcium alginate hydrogel shell. The acid resistance of the shell protected the core microspheres, thus enabling successful targeted delivery into the small intestine. Under a scanning electron microscope, the UPA microspheres exhibited wrinkled surfaces, characteristic of spherical hydrogel microspheres. Figure 4 (A). The prepared microspheres have low polydispersity (205.9 ± 8.18 μm) and a size ( Figure 4(B) A large number of microspheres were rapidly prepared using airflow control technology, with a yield of approximately 100 milligrams per minute.

[0110] The core-shell structure of the microspheres was visualized by labeling calcium alginate and uricase with rhodamine B and fluorescein isothiocyanate, respectively. Figure 4 As shown in Figure C, the Merge image simultaneously reveals the red rhodamine B-labeled calcium alginate shell and the green fluorescein isothiocyanate-labeled uricase, with uniform fluorescence distribution. Confocal laser scanning microscopy (CLSM) shows that the calcium alginate shell overlaps with the uricase, and the uricase is uniformly distributed within the calcium alginate shell.

[0111] The size of the microspheres is controllable; microspheres of different sizes can be prepared by adjusting the gas shear rate. The effect of gas flow rate on the diameter of the microspheres during hydrogel preparation was investigated. Figure 4 In the method described in section D), particle size control was achieved by adjusting the gas flow rate. Data showed that the microsphere diameter decreased significantly with increasing gas flow rate, and the microsphere diameter exhibited an almost linear correlation with the nitrogen flow rate. When the nitrogen flow rate was 0.2 L / min... -1 When the microsphere diameter is greater than 1 mm, and the flow rate is greater than 1.0 L·min⁻¹, the microsphere diameter is also greater than 1 mm. -1 At this time, the diameter of the microspheres can be less than 100 μm.

[0112] The chemical structure of the microspheres was detected using Fourier transform infrared spectroscopy (FTIR). Figure 4 (E). FTIR 3180cm -1 The increase in the peak value confirms the strong stretching vibration peak of the amide group -NH bond, proving that the microspheres contain uricase. 3400cm -1 The nearby characteristic peaks are strong-dull peaks from the stretching vibrations of the intermolecular hydrogen bonds of the -OH hydroxyl groups in dopamine, confirming the presence of dopamine in the microspheres. The results further demonstrate the successful synthesis of the microspheres, whose structural stoichiometry can be precisely controlled based on the reactant ratios.

[0113] 2. Efficient delivery of orally administered hydrogel microspheres in the small intestine

[0114] To verify that UPA microspheres can effectively reach the small intestine to release drugs and achieve good release effects, a drug release experiment using a simulated gastrointestinal fluid was designed to test the effect.

[0115] We simulated the residence time of UPA microspheres in the gastrointestinal tract after oral administration and conducted in vitro studies on the degradation of the microspheres by monitoring morphological changes during incubation in different biofluids. Figure 4(F). The diameter changes of UPA microspheres in three gastrointestinal fluids were studied. At 25 min, the diameter of UPA microspheres decreased to 41.67±7.64 mm in small intestinal fluid and to 2.51±0.51 mm in colonic fluid. After 2 hours in the gastrointestinal environment, UPA was basically decomposed and broken down in both small and colonic fluids, resulting in the complete release of uricase and dopamine. However, the diameter of the microspheres decreased to 195.3±0.58 mm in gastric fluid. Figure 4 (H). Simultaneously, we also studied the uricase release curve within the microspheres under gastrointestinal fluid conditions (H). Figure 4 (I). After treatment in simulated gastric juice (pH≈1.5) for 2–6 h, the morphology of the microspheres showed almost no significant change, with slow expansion and only 12% of uricase released into the AGF. Even after extending the treatment to 24 h, only 24% of the uricase was released into the AGF. In contrast, after suspension in simulated intestinal juice for 2 h, the UPA microspheres rapidly expanded and almost completely degraded, with 80% release after 0.5 h and complete release after 1 h. This in vitro stimulation experiment summarizes the evolution of the microspheres throughout the entire transition process in the digestive tract. The results indicate that orally administered UPA microspheres can remain intact in the stomach and, due to the abundance of proteases and alkaline conditions, gradually degrade and burst the loaded uricase and dopamine in the small intestine and colon. Figure 5 When the microspheres were incubated in simulated gastric fluid, the release of uricase and dopamine was largely preserved under the acidic environment of the stomach, while the microspheres released uricase and dopamine at a higher rate when exposed to a weakly alkaline buffer (pH 7.8). We simulated the retention time of the drug in the gastrointestinal tract after oral administration. UPA hydrogel microspheres swelled slowly in AGF, but their swelling rate was significantly higher in ASF and simulated colonic fluid (ACF). Figure 4 (J). Therefore, in vitro experiments have confirmed that calcium alginate microspheres can protect uricase and dopamine from being destroyed in the stomach, thereby inhibiting the exposure and degradation of uricase in gastric acid.

[0116] 3. In-situ formation of a polydopamine coating on the small intestinal mucosa enables efficient fixation of uricase.

[0117] After the composite microspheres disintegrated in the intestine, the hydrogen peroxide produced by uricase hydrolysis of uric acid by uricase, combined with the high concentration of catalase in the intestinal microenvironment, created a local oxidative microenvironment, enabling in-situ dopamine polymerization in the small intestinal mucosa. This in-situ immobilization of uricase in the small intestine was achieved through a polydopamine coating. This in-situ immobilization of uricase in the mucosa enabled in-situ uric acid regulation. To verify the in-situ formation of the polydopamine coating and the immobilized enzyme activity, we designed an in vitro adhesion experiment between UPA microspheres and isolated porcine small intestine. Next, we evaluated whether endogenous catalase could accelerate PDA polymerization and coating in the small intestine. The porcine gastrointestinal tract was selected as the first model tissue. Figure 6(A) is used because it shares anatomical and physiological similarities with the human digestive system. Ex vivo tissue was incubated with UPA microspheres, uric acid solution, and catalase at concentrations considered close to those found in the human intestine. Figure 6 As shown in Figure B, the porcine ileum reacted in a UPA microsphere solution, and after 3 hours, the surface of the porcine intestine was adhered with polydopamine. When the lumen surface of the small intestine was exposed to UPA microspheres and H2O2 solution, a dark brown PDA coating was observed on the epithelial surface, indicating that PDA formed in situ and deposited at the tissue anchor points due to the high reactivity of PDA. In contrast, almost no PDA was observed on the serous membrane surface of the tissue.

[0118] 4. Evaluate the effect of uric acid intervention using human intestinal fecal samples.

[0119] The human intestinal environment is complex. To verify that UPA microspheres can still regulate uric acid metabolism and decompose uric acid in the complex human intestinal system, fecal samples from gout patients were used to evaluate the effect of uric acid intervention in vitro. Human samples—fecal samples from gout patients—were obtained and mixed with UPA microspheres. The temperature and pH of the human intestinal tract were simulated as closely as possible. After reacting the microspheres with feces and catalase for 24 hours, the supernatant was centrifuged, and the remaining uric acid content was measured. The decrease in fecal uric acid concentration and the rate of decrease were observed in different patients. It was found that the in vitro fecal uric acid concentration decreased significantly in all patients. In vitro experiments on fecal samples from gout patients demonstrated that the microspheres significantly reduced fecal uric acid by up to 37%, and the breakdown was usually completed by uricase within 24 hours. Simultaneously, the effect of microspheres of different diameters on reducing fecal uric acid was studied. It was found that microspheres with a diameter of 50 micrometers to 1 millimeter had little effect on reducing uric acid under the premise of a certain drug loading. Figure 6 (E); In addition, studies on the effect of different dopamine levels on the reduction of uric acid levels in feces found that uric acid gradually decreased with increasing dopamine levels, possibly because the polymerization and immobilization of dopamine allows uricase to function for a longer period of time. Figure 6 (F). Another study investigated the effect of different uricase levels on the reduction of uric acid levels in feces, finding that uricase levels were inversely proportional to uric acid concentration. Figure 6 (G).

[0120] Furthermore, the antibacterial activity of UPA against *Escherichia coli* and *Staphylococcus aureus* was evaluated using the diffusion plate method. The similar bacterial colony counts indicated that the microspheres did not stimulate abnormal proliferation of these two common intestinal flora. Figure 7 ).

[0121] 5. In vitro anti-inflammatory effect of in-situ uricase immobilized coating

[0122] Polydopamine coatings possess antioxidant properties and also inhibit inflammatory processes. This is because ROS is consumed during the polymerization of dopamine to form PDA. Therefore, we hypothesize that this in-situ formed polydopamine coating may also have anti-inflammatory effects. In in vitro experiments, we co-incubated RAW 264.7 cells with uric acid, UA, and UPA, labeled RAW 264.7 cells with CD11, M1 macrophages with CD86, and M2 macrophages with CD206.

[0123] Uric acid, after being broken down by uricase, produces a large amount of reactive oxygen species (ROS). To verify that UPA can effectively address ROS, flow cytometry was used to quantify the distribution of macrophage subsets. The results showed a significant increase in the percentage of CD11b+ and CD206+ cells, representing M2 macrophages, from 14.85% to 26.32%, while the percentage of CD11b+ and CD86+ cells, representing M1 macrophages, significantly decreased from 38.23% to 22.15%. This substantial increase in the proportion of anti-inflammatory M2 macrophages effectively suppressed inflammation. Figure 8 (A and B in the original text). The results indicate that UPA microspheres can alleviate inflammation by promoting a shift of the immune system to an anti-inflammatory state.

[0124] Next, we attempted to explore the upstream signaling pathways mediated by UPA microspheres in the anti-inflammatory properties of Caco-2 cells. We co-incubated Caco-2 cells with uric acid, UA microspheres, and UPA microspheres. Using quantitative RT-PCR and Western blotting, we detected the mRNA expression of genes related to ABCG2 intestinal transporter, GLUT9 intestinal transporter, caspase-1, PI3K, TLR4, TLR2, TNF-α, and IL-1β in Caco-2 cells. We found that the expression of ABCG2 intestinal transporter, GLUT9 intestinal transporter, caspase-1, PI3K, TLR4, and TLR2 was upregulated in both the uric acid + UA and uric acid + UPA groups, with a more significant upregulation in the uric acid + UPA group. Meanwhile, the expression of inflammatory factors TNF-α and IL-1β was downregulated compared to the uric acid group. Figure 8(C) These phosphorylated proteins are key molecules in the ABCG2 signaling pathway, a major uric acid excretion signaling pathway in the human gut. Studies have found that reducing intestinal uric acid levels has the potential to upregulate the levels of various uric acid transporters in intestinal endothelial cells and further increase the rate of uric acid excretion from the intestinal endothelium into the intestinal lumen. We verified that the TLR4-NLRP3 inflammasome and PI3K / Akt signaling pathway in intestinal Caco-2 cells regulate the expression of soluble uric acid-stimulated PDZK1 and ABCG2 intestinal uric acid transporters in human intestinal cells. Soluble uric acid interacts with TLR2 or TLR4, initiating caspase-1 formation by recruiting the NLRP3 inflammasome composed of NLRP3, ASC, and pro-caspase-1, and activating the PI3K / Akt signaling pathway by phosphorylating Akt at Ser473 and Thr450. Activation of caspase-1 and Akt increases PDZK1 expression, thereby upregulating ABCG2 expression. Therefore, compared to regulating a single transporter protein target, regulating intestinal uric acid levels can simultaneously regulate multiple transporters, resulting in more efficient intestinal excretion of uric acid.

[0125] The results of representative Western blots of Caco-2 cell-related genes ABCG2 and GLUT9 were used to quantitatively analyze the protein levels obtained from the Western blots. Figure 8 In the D and E groups, it was found that the expression of ABCG2 and GLUT9 intestinal uric acid transporters was upregulated in both the UA and UPA groups, further verifying that multiple transporters can be simultaneously regulated by controlling intestinal uric acid levels, thereby achieving more efficient intestinal uric acid excretion.

[0126] Under LPS stimulation, inactive monocytes (M0 type) can be induced to polarize into pro-inflammatory M1 macrophages and secrete large amounts of pro-inflammatory cytokines, such as IL-1β, IL-2, and TNF-α. Synovial M1 macrophages play a crucial role in the pathogenesis of gouty arthritis and other diseases. Reducing the inflammatory expression of synovial macrophages can effectively alleviate acute gout symptoms. In the lesion microenvironment of gouty arthritis, macrophages at the local joints exhibit increased M1 polarization and release corresponding inflammatory factors, which also affects the related physiological behaviors of the affected joint. Therefore, it is necessary to comprehensively evaluate the anti-inflammatory effects of UPA by introducing co-culture conditions of macrophages and microspheres to maximize the simulation of the hyperuricemic state in vivo. We performed immunofluorescence to assess the relative mRNA expression of M1 and M2 macrophage-related genes (…). Figure 9In comparisons A and B, it was found that the UPA microsphere group, due to reduced ROS production, showed a decrease in pro-inflammatory M1 macrophages relative to the UA group, while an increase in anti-inflammatory M2 macrophages. In the UA group, pro-inflammatory M1 macrophages increased, and anti-inflammatory M2 macrophages increased, but not as much as in the UPA group. Semi-quantitative CD86 and CD206 fluorescence results showed that the number of M1 macrophages in the uric acid + UA group was significantly higher than that in the uric acid + UPA group and the uric acid group; the number of M2 macrophages in the uric acid + UPA group was significantly higher than that in the uric acid group and the uric acid + UA group. Figure 9 (C and D in the middle).

[0127] To further elucidate the anti-inflammatory mechanism of UPA microspheres, the inhibitory effect of UPA on M1 polarized macrophages was assessed by qPCR after detecting the mRNA levels of IL-1β and TNF-α. First, LPS induced RAW 264.7 cells (M0 type) to M1 type; compared with M0 type macrophages, M1 type macrophages expressed significantly increased inflammatory cytokines, such as... Figure 9 As shown in Figures E and F. After incubation with uric acid, UA microspheres, and UPA microspheres, the expression levels of IL-1β and TNF-α mRNA in M1 macrophages were significantly reduced (P<0.01), and the expression levels of IL-1β and TNF-α mRNA in UPA were also significantly reduced (P<0.01). The UPA group was closest to the M0 control group. This indicates that the UPA microsphere group had the strongest inhibitory effect on inflammatory expression in M1 macrophages. As expected, compared with uricase, dopamine reduced the production of reactive oxygen species (ROS) through an enzyme cascade reaction, inhibited mRNA expression, and suppressed the production of IL-1β and TNF-α, demonstrating excellent anti-inflammatory capabilities. The uric acid + UA group produced significantly more ROS than the uric acid + UPA group and the uric acid group. In the in vivo inflammatory environment, the M1 / M2 balance tilts towards M1, and increased secretion of pro-inflammatory factors recruits more macrophages, inducing high levels of matrix metalloproteinases (MMPs), thereby affecting the proliferation and invasion of synovial fibroblasts. Therefore, inhibiting M1 polarization and inflammatory cytokine secretion caused by sodium urate crystals in the joint is one of the strategies for treating gout. Numerous studies have reported that local injection of uricase into the joint significantly increases the expression of inflammatory factors, especially TNF-α, in M1-polarized macrophages. The main mechanism is that uricase breaks down uric acid, producing a large amount of free reactive oxygen species, which in turn upregulates the expression levels of inflammatory factors IL-1β and TNF-α mRNA.

[0128] 6. Cytotoxicity and biocompatibility

[0129] To examine the biocompatibility of the microspheres with the human body and whether they exhibit cytotoxicity, we conducted biocompatibility experiments using hydrogel microsphere extracts to ensure that the same number of microspheres were co-cultured with cells. After co-culturing intestinal epithelial cell lines Caco-2 and RAW 264.7 with different concentrations of hydrogel microsphere extracts for 1, 2, and 3 days, we used qualitative and quantitative methods to study the biocompatibility of the microspheres. Based on Live / Dead staining data, the Uricase, UA, and UPA microsphere extract groups showed similar biocompatibility to the control group. Figure 10 (A and B). The UPA extract group maintained good biocompatibility for 3 days, as reported by the Cell Counting Kit 8 (CCK-8) data. Figure 11 ).

[0130] Furthermore, we monitored intracellular reactive oxygen species (ROS) levels in RAW 264.7 cells using 2',7'-dichlorofluorescein diacetate (DCFH-DA), a non-fluorescent reagent (DCF) that reacts with intracellular ROS to produce the fluorescence of 2,7-dichlorofluorescein. The fluorescence intensity of DCF correlated with the intracellular ROS content. Figure 10 As shown in Figure C, uricase-treated cells exhibited significant green fluorescence after the addition of UA, indicating a large amount of ROS production. Meanwhile, RAW 264.7 cells treated with UPA showed negligible green fluorescence, suggesting that UPA significantly reduces the impact of ROS. Simultaneously, semi-quantitative fluorescence results showed (…). Figure 10 Compared to the uricase group, the UPA group significantly reduced ROS production. These results, including ROS levels in RAW 264.7 cells, strongly suggest that our designed UPA has minimal toxicity for the treatment of hyperuricemia.

[0131] 7. Evaluation of treatment efficacy in animal models of hyperuricemia

[0132] The above studies examined the anti-inflammatory activity and in vitro antibacterial potential of UPA, as well as its mucosal adhesion ability in the ileal mucosa. Therefore, it is necessary to conduct in vivo studies to evaluate the therapeutic effect of UPA on mouse models of hyperuricemia. Figure 12Hypoxanthine- and potassium oxychloride-induced hyperuricemia (HUA) is one of the most commonly used animal models of hyperuricemia (HUA) and has been introduced to simulate HUA. The hyperuricemia model was established in Balb / c mice and divided into the following five groups (n=7 per group): (1) normal group as negative control group, (2) hyperuricemia mouse model group without treatment as positive control group, (3) hyperuricemia mouse model group + uricase group, (4) hyperuricemia mouse model + uricase@Alg-Ca, (5) hyperuricemia mouse model + UPA. Blood samples (50 μl) were collected from the orbital cavity at specific time intervals, such as 1, 3, 16 and 24 hours, and the serum UA concentration was measured by a uric acid assay kit. Mice treated with UPA showed the lowest uric acid (UA) levels, almost reaching normal levels. The UPA group had a blood uric acid concentration of 101.9 ± 27.71 mmol / L, which was closer to the Sham group's level of 79.6 ± 4.99 mmol / L, and significantly better than the HUA group's 355.2 ± 60.78 mmol / L, the Uricase group's 353.1 ± 59.58 mmol / L, and the UPA group's 145.5 ± 24.69 mmol / L. During treatment, changes in mouse body weight, water intake, urine, and fecal uric acid levels were monitored. There was no significant change in body weight between the UPA group and the normal group, but the urinary uric acid level decreased significantly by 20.4% compared to the HUA group. Figure 12 In the middle D), the level of uric acid in feces increased significantly, effectively increasing fecal uric acid excretion by 27.6% and reducing blood uric acid by 71%. Figure 12 (F). In summary, the above results demonstrate that the enzyme cascade reaction based on UPA microspheres has a significant therapeutic effect on hyperuricemic mice, showing its potential for clinical treatment of hyperuricemia.

[0133] After confirming the uric acid-lowering effect of UPA microspheres, further studies were conducted to verify the regulation of the immune response and safety in a hyperuricemia model. To detect the activation pathway of intestinal transport proteins in mice, qRT-PCR analysis was performed using mouse ileum tissue. Compared with the Sham and HUA groups, the mRNA levels of pro-inflammatory cytokines (TNF-α and IL-1β) were significantly reduced in mice treated with UPA microspheres. Figure 12 In the Uricase@Alg-Ca group and the UPA group, the expression of ABCG2, GLUT9, Caspase-1, PI3K, TLR4, and TLR2 was upregulated.

[0134] Next, we attempted to explore the upstream signaling pathway of uric acid transport mediated by UPA-mediated ileal transport proteins in mice. We used quantitative RT-PCR to detect the mRNA expression of genes related to ABCG2, GLUT9, caspase-1, PI3K, TLR4, TLR2, TNF-α, and IL-1β in ileal tissue. We found that the expression of ABCG2, GLUT9, caspase-1, PI3K, TLR4, and TLR2 was upregulated in both the UA and UPA groups, while the expression of inflammatory factors TNF-α and IL-1β was downregulated. These phosphorylated proteins are key molecules in the ABCG2 and GLUT9 signaling pathways, which are major uric acid excretion signaling pathways in the human gut. This study suggests that reducing intestinal uric acid levels has the potential to upregulate the levels of various uric acid transport proteins in intestinal endothelial cells and further increase the rate of uric acid excretion from the intestinal endothelium into the intestinal lumen. We validated that the TLR4-NLRP3 inflammasome and PI3K / Akt signaling pathway in intestinal cells regulate the expression of PDZK1 and ABCG2 intestinal uric acid transporters stimulated by soluble uric acid in human intestinal cells. Soluble uric acid interacts with TLR2 or TLR4, initiating caspase-1 formation by recruiting the NLRP3 inflammasome composed of NLRP3, ASC, and pro-caspase-1, and activating the PI3K / Akt signaling pathway through phosphorylation of Akt at Ser473 and Thr450. Activation of caspase-1 and Akt increases PDZK1 expression, thereby upregulating ABCG2 expression. Therefore, compared to the regulation of a single transporter target, regulating intestinal uric acid levels can achieve simultaneous regulation of multiple transporters, resulting in more efficient intestinal uric acid excretion.

[0135] The weight of the UPA group was 18.67±0.02g, slightly lower than the Sham group (18.7±0.02g), better than the UA group (18.6±0.01g), and significantly higher than the HUA group (18.16±0.05g) and the Uricase group (18.22±0.08g). Statistical data show that the UPA group had a significantly greater weight than the HUA group (p<0.0001). Figure 12 The H group also had lower water intake; the average daily water intake of the UPA group was 7.06±0.16g, slightly lower than that of the HUA group (7.67±0.19g). This is because mice with hyperuricemia often feel thirsty and thus increase their water intake. Figure 12 The results (G) indicate that the mice in the UPA group had milder symptoms of hyperuricemia, all of which suggest that UPA significantly protects mice from the effects of hyperuricemia.

[0136] In addition, edema and gait analysis were performed at 6 and 24 hours to monitor the degree of pain in the affected limb. Figure 13Mice with red forepaws and blue hind paws were allowed to walk freely from side to side on a 100cm × 10cm track. Mouse footprints were recorded, and gait analysis during the walking process clearly revealed pain in the affected limbs. In the normal group, the forepaw and hind paw imprints overlapped relatively, while in the MSU group, the forepaw and hind paw imprints were significantly separated. Oral administration of UPA microspheres reduced this tendency and partially restored movement at 6 and 24 hours. The average step length in the UPA group at 6 hours was approximately 3.81 ± 0.09 cm, close to that of the Sham group (3.97 ± 0.15 cm), and better than the UA group (3.66 ± 0.06 cm), the uricase group (2.40 ± 0.02 cm), and the MSU group (2.14 ± 0.15 cm). At 24 hours, the average step length in the UPA group was approximately 3.66 ± 0.05 cm, more similar to that of the Sham group (4.01 ± 0.01 cm), and better than the UA group (3.33 ± 0.07 cm), the uricase group (2.09 ± 0.13 cm), and the MSU group (1.96 ± 0.06 cm). Generally, the gait analysis further illustrates that uricase alleviates pain caused by MSU deposition.

[0137] Micro-CT and H&E staining of the ankle joint showed sodium urate crystal deposition in the MSU group, confirming the successful establishment of the acute gouty arthritis mouse model. The ankle joints of the UPA group were similar to those of the normal group. Figure 13 (AB). In mice treated with MSU, MSU-induced gouty arthritis characterized by bone deformities, severe joint erosion, and associated soft tissue swelling was observed. Both the UA microsphere group and the UPA microsphere group showed significant improvement in paw swelling and bone destruction after treatment.

[0138] Mice were euthanized, and their intestines were harvested, frozen, ground, and centrifuged. The supernatant was then collected to measure the H2O2 content within the mouse intestines. Changes in H2O2 concentration within the mouse intestines revealed… Figure 13 In the C group, the amount of reactive oxygen species produced by UPA was significantly reduced compared to that of the UA group, effectively solving the problem of a large amount of H2O2 produced during the decomposition of uric acid by uricase.

[0139] To verify in vivo mucosal adhesion properties, mice were orally administered UPA (made from fluorescein isothiocyanate-labeled uricase) and FITC-labeled uricase, and observed under IVIS. The intestinal tracts of mice in the 1-hour and 4-hour groups were observed. Figure 13 The results (D) showed a stronger fluorescence signal in gout mice, indicating a prolonged fluorescence residence time of FITC-UPA in the intestine, which verifies the mucosal adhesion properties of UPA microspheres in vivo. These results can be attributed to enhanced retention of dopamine microspheres in the intestine.

[0140] according to Figure 14Colonic injury scores stained with hematoxylin and eosin (H&E) in groups A and B (p<0.001) showed that UPA treatment also helped maintain the integrity of the ileal epithelium and reduce the infiltration of pro-inflammatory cells in the mucosa. Evidence shown in mice suggests that UPA is a promising therapy. Furthermore, we compared the therapeutic effects of UPA and UA. The study found that UPA was significantly superior to the UA group in reducing hyperuricemia, both in terms of body weight and ileal injury score, while oral uricase solution showed similar therapeutic effects compared to HUA. Figure 14 (E). The difference in treatment efficacy between the UPA microsphere group and the UA microsphere group may be due to the PDA immobilizing uricase to the ileal wall during delivery, which exerts a lasting effect and better activates intestinal transport proteins to transport uric acid.

[0141] Histological evaluation was performed on the ileum tissues of mice from each group. Compared with the MSU group, the intestinal tissue damage in the UPA treatment group was significantly reduced, while the structural integrity of the intestinal villi, intestinal mucosa, and serosa was well preserved. Figure 14 (A and B, p<0.0001). This result further confirms that UPA microspheres can effectively inhibit the inflammatory response of uric acid in the intestine.

[0142] We then conducted in-depth analysis to understand how UPA microspheres protected mice from the effects of HUA. In the ileum tissue of the HUA group mice, the proportions of ABCG2 and GLUT9 transporter activities were significantly increased in the UPA group. Interestingly, we also observed that epithelial cells remained relatively intact after UPA administration. Therefore, we used Western blotting to detect the protein levels of ABCG2 and GLUT9 transporters in the ileum tissue. We found that UPA administration significantly upregulated the expression of ABCG2 and GLUT9 transporters. Western blotting analysis of samples from the upper, middle, and lower ileum segments of the Sham, HUA, and UPA groups revealed no significant differences in the upper, middle, and lower ileum segments among the groups, but the expression of ABCG2 and GLUT9 transporters in the ileum segment of the UPA group was significantly higher than that in the ileum segments of the Sham and HUA groups. Figure 14 (I, J)

[0143] In addition, we performed H&E staining analysis on the major organs of mice in each group to observe the toxic effects of the microspheres on the mice's systemic organs. The tissues were fixed in paraformaldehyde and embedded in paraffin. Tissue sections stained with hematoxylin and eosin (H&E) are shown below. Figure 15 As shown, compared with the healthy group, no significant toxic effects were observed in the major organs such as the heart, liver, spleen, lungs, and kidneys.

[0144] 8. Evaluation of treatment efficacy in animal models of acute gouty arthritis

[0145] In addition to symptoms of hyperuricemia, gout patients often present with symptoms of acute gouty arthritis. Therefore, it is necessary to conduct in vivo studies to evaluate the therapeutic effect of UPA microspheres on a mouse model of acute gouty arthritis. We prepared sodium urate (MSU) crystals and then injected the synthesized MSU into the right ankle joint of Balb / c mice to establish an acute gout model. Figure 16 (A). Then, physiological saline was injected into the joint cavity of the normal group mice. The mice were divided into the following five groups (n=7 in each group): (1) the normal group was the negative control group, (2) the gouty arthritis mouse model group was not treated and was the positive control group, (3) the MSU group + Uricase group, (4) the MSU + UA group, and (5) the MSU + UPA group were injected into the joint cavity with MSU (50μL).

[0146] Except for the UPA group, arthritis scores increased rapidly with disease progression in all mice, while disease progression was significantly suppressed in the UPA group, resulting in lower arthritis scores than the other groups. We measured the ankle joint diameter of the affected limb (…). Figure 16 (B) To assess joint edema after injection. Results showed that 6 hours after MSU injection into the ankle joint, the average ankle joint diameter in the oral UPA microsphere group was 2.64±0.12 mm, close to the Sham group (2.05±0.05 mm), better than the UA group (3.55±0.05 mm), and significantly different from the average ankle joint diameters of the MSU group (4.17±0.06 mm) and the Uricase group (4.05±0.06 cm). 24 hours after MSU injection into the ankle joint, the average ankle joint diameter in the oral UPA microsphere group was 2.44±0.14 mm, close to the Sham group (2.05±0.05 mm), better than the UA group (2.77±0.16 mm), and significantly different from the average ankle joint diameters of the MSU group (3.33±0.06 mm) and the Uricase group (3.31±0.11 cm). Figure 16 As shown in Figure C, mice in the control group and those injected directly into the joint showed significant paw erythema and swelling. Conversely, these signs were significantly reduced in mice administered UPA. In gouty arthritis, cellular infiltration is often associated with disease progression and tissue remodeling. Hematologic and epithelial analysis (H&E) of synovial tissue was performed to assess inflammatory cell recruitment after MSU injection. The results showed that ankle swelling was most severe 6 hours after injection, followed by gradual relief. The UPA microsphere group exhibited the least ankle swelling, most closely resembling the normal group, and significantly better than the MSU group, validating that UPA microspheres significantly alleviate joint swelling and synovial inflammation during acute gout.

[0147] like Figure 16As shown in Figure D, long-term oral administration of microspheres reduced MSU-induced inflammatory cell infiltration into the synovium. Histopathologically, injection of sodium urate crystals revealed significant inflammatory cell infiltration in the synovium compared to normal controls. Treatment interventions with UA and UPA reduced the inflammatory response, with relatively fewer inflammatory cells observed in both groups. Furthermore, UPA was more effective than UA, indicating that polydopamine immobilizes uricase, prolonging its duration of action and reducing inflammatory cell swelling.

[0148] (III) Conclusion

[0149] This invention utilizes airflow control and ion crosslinking technologies to construct intestinal "bursting" oral hydrogel microspheres loaded with uricase, achieving immobilization of uricase in the intestinal mucosa and thus, for the first time, inhibiting gout by excreting free uric acid through the intestine. A small amount of uricase upregulates the expression of uric acid transport proteins in intestinal endothelial cells, improving the ability to excrete uric acid from endothelial cells into the intestine and the efficiency of excretion through feces. In vitro experiments and in vivo experiments in mice have shown that the intestinal "bursting" hydrogel microspheres achieve precise intestinal delivery of dopamine and uricase. In vitro experiments using fecal samples from clinical gout patients and pig small intestines have demonstrated that the microspheres significantly reduce fecal uric acid by up to 37%. Experiments in mouse models of hyperuricemia and acute gouty arthritis have confirmed that the microspheres effectively target intestinal uric acid excretion in hyperuricemic mice and arthritic joints in mice with gouty arthritis, effectively increasing fecal uric acid excretion by up to 27.6% and reducing serum uric acid by up to 71%. Furthermore, 16S ribosomal RNA sequencing revealed that the microspheres optimized the gut microbiota composition, significantly increasing probiotics such as *Muciniphila* and *Verrucomicrobiales*, while inhibiting harmful bacteria commonly found in the gut of gout patients, such as *Bacteroidetes*, thereby improving gut homeostasis. In summary, we have achieved, for the first time, the treatment of gout through uric acid excretion via the small intestinal mucosa.

[0150] Comparative Example 1

[0151] Effects of different microsphere sizes, different uricase concentrations, and dopamine concentrations on intestinal uric acid excretion (see Tables 2-4):

[0152] Small intestinal tissue from pigs was obtained from the slaughterhouse. Pigs were euthanized, and fresh tissue was removed and stored on ice. Tissue (4 cm²) was exposed to uric acid and microsphere solution (10 mL), and washed three times with PBS buffer (1X) to remove excess PDA. Microspheres were placed in environments containing uric acid, artificial small intestinal fluid, and isolated pig small intestine, and the residual uric acid in the pig small intestine was studied at different pH solutions and time points.

[0153] We designed microspheres of different sizes and tested their effect on degrading uric acid in a simulated human environment during an in vitro porcine small intestine adhesion experiment. The microsphere sizes were set at 200 μm, 400 μm, 600 μm, 1 mm, 2 mm, and 3 mm, as shown in Table 2. We found that different microsphere sizes had little effect on uric acid degradation; comparatively, smaller microspheres showed better uric acid reduction. We also set up control groups 1 and 2, one without uricase and the other without dopamine, to examine the effect of microspheres on uric acid degradation. The results showed that without uricase, the microspheres could not degrade uric acid; without dopamine, the microspheres could only degrade a small portion of the uric acid, leaving a large portion remaining, and the uricase activity was also low.

[0154] Meanwhile, we designed microspheres containing different concentrations of uricase, as shown in Table 3. We found that microspheres containing 10 U / mg uricase had the best effect on degrading uric acid.

[0155] In addition, we designed microspheres containing different concentrations of dopamine, as shown in Table 4. We found that microspheres containing 0.01 g / ml dopamine had the best effect on degrading uric acid.

[0156] Table 2

[0157]

[0158] Table 3

[0159]

[0160] Table 4

[0161]

[0162] In addition, the effects of hydrogel size and microsphere yield on sodium alginate solution flow rate were investigated, and a suitable flow rate and ratio were finally found to ensure that the size is stably controlled at 180-220 micrometers while achieving high yield.

[0163] The relationship between yield and size at different flow rates was investigated. Particle size control was achieved by adjusting the flow rate of the sodium alginate solution. As the flow rate of the sodium alginate solution increased, the microsphere diameter decreased significantly, and the microsphere diameter showed a nearly linear relationship with the flow rate of the sodium alginate solution (e.g., ...). Figure 17 When the sodium alginate solution flow rate is 5 ml / h, the microsphere diameter is greater than 1 mm; when the sodium alginate solution flow rate is greater than 30 ml / h, the microsphere diameter is less than 100 μm. When the sodium alginate solution flow rate is 20-25 ml / h, the size can be effectively controlled at around 200 μm, while the yield is controlled at 100 mg per minute.

Claims

1. A method for preparing oral hydrogel microspheres that target the intestines to inhibit gout, characterized in that, Includes the following steps: (1) Dissolve uricase and dopamine in sodium alginate solution, wherein the concentration of sodium alginate is 1% w / v, the concentration of uricase is controlled at 10-20 U / mg, and the concentration of dopamine is 0.01-0.1 g / ml, and disperse by ultrasonication; (2) Place the solution obtained in step (1) in a microfluidic device and use gas microfluidic technology to shear the solution into uniform droplets under the action of nitrogen gas flow. Control the flow rate of nitrogen gas to 0.2-1.0 L / min and control the flow rate of the solution obtained in step (1) to 10-30 mL / h. (3) Drop the liquid obtained in step (2) into a calcium chloride solution with a concentration of 100 mM, and cross-link it to form the oral hydrogel microspheres that target the intestine to inhibit gout.

2. The preparation method according to claim 1, characterized in that, In step (1), the concentration of uricase is controlled to be 10 U / mg.

3. The preparation method according to claim 1, characterized in that, In step (1), the concentration of dopamine is controlled to be 0.01 g / ml.

4. The preparation method according to claim 1, characterized in that, In step (2), the flow rate of nitrogen is controlled to be 0.8 L / min.

5. The preparation method according to claim 1, characterized in that, In step (2), the flow rate of the solution obtained in step (1) is controlled to be 20-25 mL / h.

6. Oral hydrogel microspheres for targeting intestinal inhibition of gout, prepared by the method according to any one of claims 1-5.

7. The oral hydrogel microspheres for targeted intestinal inhibition of gout according to claim 6, characterized in that, The diameter of the hydrogel microspheres is 180-220 micrometers.

8. The use of the oral hydrogel microspheres for targeting intestinal inhibition of gout as described in claim 6 in the preparation of a medicament for treating gout.

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