Selenized magnetic porous carbon microspheres for blood perfusion as well as preparation method and application of selenized magnetic porous carbon microspheres

Abstract

The preparation method comprises the following steps: by taking traditional Chinese medicine puffball as a carbon source, carrying out iron ion complexing modification to form a metalloid organic complex material, then carrying out high-temperature calcination in an inert atmosphere to prepare the magnetic porous carbon microspheres, and then sequentially carrying out KOH and selenium powder activation treatment to obtain the selenized magnetic porous carbon microspheres for blood perfusion. And preparing the selenized magnetic porous carbon microspheres with high specific surface area, large pore capacity and hierarchical pore structure. The raw materials used in the invention are low in cost, green and environment-friendly, the preparation method is simple, the conditions are mild, and the obtained selenized magnetic porous carbon microspheres are stable in performance, have biocompatibility, self-antibiosis, uricase-like and antioxidant enzyme-like functions, and are expected to be used as a blood perfusion adsorbent for in vitro treatment of refractory acute gout kidney or renal failure.

Description

Technical Field

[0001] The present invention belongs to the technical field of blood perfusion adsorbents, and specifically relates to a method for preparing selenized magnetic porous carbon microspheres by using a traditional Chinese medicine puffball as a carbon source and a spherical self-template, combined with a metal chelation-assisted self-assembly and an inert atmosphere high-temperature-assisted KOH-selenium powder activation strategy, as well as the application of the material in the field of blood purification for the treatment of refractory acute gouty kidney or renal failure. Background Art

[0002] Gouty nephropathy is a metabolic disease caused by the accumulation of uric acid in the body, resulting in the formation of urate crystals that are deposited in the kidneys. In recent years, the number of patients with gouty nephropathy has increased with the increasing incidence of hyperuricemia and gout. Uric acid is primarily excreted through the kidneys, and renal impairment can inhibit uric acid excretion. Untreated, this can lead to renal failure.

[0003] The primary treatment for relieving gouty kidney disease is to control uric acid levels. Currently, uric acid-lowering drugs are divided into three main categories: uric acid production inhibitors, uricosuric agents, and uricosuric acid oxidase drugs. Clinical data show that while these drugs are effective in lowering uric acid, they also have numerous side effects. For example, allopurinol can cause hypersensitivity reactions and can lead to toxic accumulation in patients with renal insufficiency; febuxostat has potential cardiovascular toxicity; and benzbromarone is hepatotoxic and can cause liver failure, resulting in its withdrawal from the market in Japan and the United States. Uricase degrades uric acid, converting it into soluble allantoin, and is widely used to treat hyperuricemia or gout. Compared to traditional chemotherapy, uricase preparations offer significant advantages in lowering uric acid levels and dissolving urate crystals. Uricase is characterized by rapid uric acid reduction and wide applicability, and uricase drugs are particularly effective in patients with acute and refractory gouty kidney disease. However, as a protein drug administered by injection, uricase has the pain points of high price and easy inactivation. After injection, it may cause side effects such as allergic reactions, nausea, and vomiting. In particular, uricase catalyzes uric acid to produce hydrogen peroxide byproducts, which further increases the burden of oxidative stress. It is worth noting that in cases where traditional drug treatment is ineffective and acute gouty kidney is accompanied by organ failure, there is still a lack of effective treatment methods. Therefore, there is an urgent need for a new drug or new method to treat refractory gouty kidney in clinical practice to fill the bottleneck problem of existing drug incurable diseases.

[0004] Hemoperfusion is a crucial component of blood purification, removing endogenous and exogenous toxins through adsorption. Hemoperfusion has played a crucial role in the adjuvant treatment of conditions such as acute poisoning, hepatorenal failure, and sepsis. Clinical practice has demonstrated that hemoperfusion, as a novel therapy, has shown potential for clearing uric acid from the blood and alleviating acute gouty kidney disease. Adsorbents are a core component of the hemoperfusion system and a key component in determining the body's ability to clear toxins. In recent years, a variety of novel hemoperfusion adsorbent materials have been developed, including carbon-based materials, polymer-based materials, magnetic nanoparticles, composite materials, and metal-organic frameworks. However, existing adsorbents still suffer from numerous drawbacks, including poor biocompatibility, limited adsorption selectivity, low mechanical strength, low uric acid removal efficiency, high coagulation risk, limited functionality, high treatment costs, and the lack of oxidative stress regulation. In contrast, magnetic materials can minimize adsorbent infiltration into the bloodstream during hemoperfusion, thereby reducing the risk of embolism. Given the limitations of existing adsorbents, the development of low-cost, multifunctional magnetic hemoperfusion adsorbents is crucial.

[0005] Among the many hemoperfusion materials, carbon-based materials have attracted much attention in the field of blood purification due to their advantages of good biocompatibility and excellent adsorption performance. Activated carbon has been used in the field of clinical hemoperfusion for many years, but clinical carbon-based adsorbents have the disadvantages of single function, high risk of embolism, poor renewability and insufficient detoxification performance, which greatly limits their effectiveness in the clinical treatment of critical and severe diseases. Existing clinical activated carbon used for hemoperfusion mainly achieves auxiliary treatment of gouty kidney by adsorbing urate, but has almost no self-antibacterial, oxidative stress regulation and uricase-like functions, which greatly limits its detoxification effect in hemoperfusion treatment of acute gouty kidney. Therefore, the development of magnetic carbon-based hemoperfusion adsorbents with good biocompatibility, low cost and diversified functions has important clinical value in improving the treatment effect and treatment cost of acute gouty kidney or refractory gouty kidney. Summary of the Invention

[0006] To address the bottlenecks of clinical activated carbon used in hemoperfusion, such as poor detoxification performance, lack of oxidative stress regulation, and lack of self-antibacterial and uricase-like functions, the present invention provides selenized magnetic porous carbon microspheres with uricase-like, antioxidant-like, and antibacterial functions. Leveraging their excellent self-antibacterial, adsorption, and multi-enzyme catalytic activities, they are expected to achieve the goal of synergistic hemoperfusion treatment of acute gouty kidney. This not only provides a new option for the development of low-cost, self-antibacterial, biocompatible hemoperfusion adsorbents that integrate oxidative stress regulation and uricase-like functions, but also provides a new paradigm for constructing an integrated "adsorption-catalysis-oxidative stress regulation" hemoperfusion treatment for acute gouty kidney.

[0007] In order to achieve the above object, the present invention uses the Chinese herbal medicine puffball as a carbon source, adopts a technology combining metal chelation-assisted self-assembly, inert atmosphere high temperature cracking and KOH-Se powder activation method to prepare selenized magnetic porous carbon microspheres (denoted as Se-KOH-MPCM), and the specific preparation method is prepared by the following steps:

[0008] Step 1: Peel the puffball, take the inner fluff, wash and dry it to obtain puffball powder.

[0009] Step 2: Dissolve the iron salt in distilled water, add the puffball powder from step 1, stir at room temperature for 2 to 4 hours, and then heat-induce the assembly reaction at 60 to 90° C. for 12 to 48 hours.

[0010] Step 3: calcining the product after the reaction in step 2 at 550-700° C. for 1-3 hours under inert gas protection, washing with distilled water and anhydrous ethanol in sequence, and drying to obtain puffball-derived magnetic carbon microspheres.

[0011] Step 4: The puffball-derived magnetic carbon microspheres in step 3 are mixed with KOH in a mass ratio of 1:0.1 to 1:3, calcined at 500 to 800° C. for 1 to 3 hours under inert gas protection, washed with distilled water and anhydrous ethanol in turn, and dried to obtain KOH-activated magnetic porous carbon microspheres.

[0012] Step 5: The KOH-activated magnetic porous carbon microspheres in step 4 are mixed with selenium powder in a mass ratio of 1:0.2 to 1:2, calcined at 550 to 700° C. for 1 to 3 hours under inert gas protection, washed with distilled water and anhydrous ethanol in turn, and dried to obtain selenized magnetic porous carbon microspheres.

[0013] Furthermore, in the above step 2, the mass ratio of iron (III) in the iron salt to puffball powder is preferably 1:3 to 1:9; and the concentration of the iron salt added to the distilled water is preferably 0.6 to 1.8 mol / L.

[0014] Furthermore, in the above step 2, the iron salt is any one of ammonium ferric citrate, anhydrous ferric chloride, ferric ammonium oxalate, and ferric nitrate nonahydrate.

[0015] Furthermore, in the above step 2, the assembly reaction is preferably heat-induced at 70° C. for 24 hours.

[0016] Furthermore, in the above step 3, the product after the reaction in step 2 is preferably calcined at 600° C. for 2 hours under the protection of inert gas.

[0017] Furthermore, in the above step 4, the puffball-derived magnetic carbon microspheres in step 3 are preferably mixed with KOH in a mass ratio of 1:2, and calcined at 700° C. for 1.5 hours under inert gas protection.

[0018] Furthermore, in the above step 5, the KOH-activated magnetic porous carbon microspheres in step 4 are preferably mixed with selenium powder in a mass ratio of 1:0.5, and calcined at 600° C. for 2 hours under inert gas protection.

[0019] In steps 3, 4, and 5 above, the inert gas is nitrogen or argon.

[0020] The present invention also provides use of the selenized magnetic porous carbon microspheres as an adsorbent in blood perfusion.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] 1. The present invention uses puffball as raw material, soaks the washed, dried, peeled puffball powder in an aqueous solution of iron salt, and through impregnation and heat-induced assembly, promotes the coordination of iron ions to modify the puffball to form a metal-like organic conjugated complex material. At the same time, the inherent microspherical structure of the puffball powder serves as a self-template, and the iron salt serves as a pore regulator and magnetic source during the high-temperature cracking process. Through high-temperature carbonization with inert gas, KOH chemical activation and selenium powder in-situ activation treatment, a series of selenized magnetic porous carbon microspheres with high specific surface area, large pore capacity and multi-level pore structure are prepared. The selenized magnetic porous carbon microspheres of the present invention not only have the advantages of good biocompatibility, self-antibacterial and removal of uric acid and other liver and kidney toxins, but also have good uricase-like and antioxidant enzyme-like catalytic activity, which provides a new idea for realizing the "adsorption-catalysis-oxidative stress regulation" type blood perfusion treatment for acute gouty kidney.

[0023] 2. The present invention uses the traditional Chinese medicine puffball to replace toxic chemical reagents to prepare selenized magnetic porous carbon microspheres for blood perfusion, which can reduce the manufacturing cost of microspherical carbon materials. The preparation method is simple, the conditions are mild, and large-scale production can be achieved. The obtained selenized magnetic porous carbon microspheres have stable performance and are expected to be used as a new generation of carbon-based blood perfusion adsorbents for the auxiliary treatment of acute gouty kidney or refractory hyperuricemia.

[0024] 3. The selenized magnetic porous carbon microspheres of the present invention can regulate the blood perfusion detoxification performance through the external magnetic field, and their inherent magnetic separation performance can maximize the reduction of thrombosis risk. In particular, the introduction of selenium, an essential element for the human body, can regulate the oxidative stress function of the blood microenvironment. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a scanning electron microscope image of Se-KOH-MPCM prepared in Example 1.

[0026] Figure 2 is the X-ray powder diffraction pattern of Se-KOH-MPCM prepared in Example 1.

[0027] Figure 3 This is the hysteresis curve of Se-KOH-MPCM prepared in Example 1.

[0028] Figure 4 This is a diagram showing the effect of Se-KOH-MPCM prepared in Example 1 and the control carbon material in removing uric acid.

[0029] Figure 5 This is a diagram showing the effect of Se-KOH-MPCM prepared in Example 1 in clearing uric acid in solutions with different pH values.

[0030] Figure 6 This is a diagram showing the effect of Se-KOH-MPCM prepared in Example 1 on clearing uric acid in uric acid solutions of different concentrations over time.

[0031] Figure 7 This is a graph verifying the uricase-like activity of Se-KOH-MPCM prepared in Example 1 and the control material.

[0032] Figure 8 This is a graph showing the oxygen consumption changes of Se-KOH-MPCM prepared in Example 1 in the degradation of uric acid.

[0033] Figure 9 This is a diagram showing the scavenging effect of Se-KOH-MPCM prepared in Example 1 and the control carbon material on scavenging DPPH free radicals.

[0034] Figure 10 This is a diagram showing the scavenging effect of Se-KOH-MPCM prepared in Example 1 and the control carbon material on scavenging ABTS free radicals.

[0035] Figure 11 This is a diagram showing the scavenging effect of Se-KOH-MPCM prepared in Example 1 and the control carbon material on scavenging superoxide radicals.

[0036] Figure 12 This is the oxygen production curve of Se-KOH-MPCM prepared in Example 1 and the control carbon material in removing hydrogen peroxide.

[0037] Figure 13 This is a diagram showing the antibacterial effect of Se-KOH-MPCM of different concentrations prepared in Example 1 on Escherichia coli (the inset is a real antibacterial image).

[0038] Figure 14 This is a diagram showing the antibacterial effect of Se-KOH-MPCM of different concentrations prepared in Example 1 on Staphylococcus aureus (the inset is a real antibacterial image).

[0039] Figure 15 This is a diagram showing the adsorption effect of Se-KOH-MPCM prepared in Example 1 on common antibiotics.

[0040] Figure 16 This is a diagram showing the adsorption effect of Se-KOH-MPCM prepared in Example 1 on hepatotoxins.

[0041] Figure 17 Graph showing the hemolytic performance of Se-KOH-MPCM prepared in Example 1 at different concentrations.

[0042] Figure 18 This is a diagram showing the effect of Se-KOH-MPCM prepared in Example 1 on clearing uric acid from a pig blood system under dynamic blood perfusion conditions.

[0043] Figure 19 This is a diagram showing the effect of Se-KOH-MPCM prepared in Example 1 on clearing creatinine from a pig blood system under dynamic hemoperfusion conditions. DETAILED DESCRIPTION

[0044] The preferred embodiments of the present invention are described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0045] Example 1

[0046] The preparation method of selenized magnetic porous carbon microspheres provided in this embodiment includes the following steps:

[0047] Step 1: Peel the puffball, take the inner fluff, wash and dry it to obtain puffball powder.

[0048] Step 2: Disperse 15 g of puffball powder into 50 mL of 0.6 mol / L ammonium ferric citrate aqueous solution, mechanically stir at room temperature for 3 hours, and then heat-induce the assembly reaction at 70°C for 24 hours.

[0049] Step 3: The product after the thermal induced assembly reaction in step 2 was calcined at 600°C for 2 hours under nitrogen protection, washed with distilled water and anhydrous ethanol in sequence after cooling, and dried at 70°C to obtain puffball-derived magnetic carbon microspheres (denoted as MPCM).

[0050] Step 4: The MPCM obtained in step 3 was mixed with KOH in a mass ratio of 1:2, calcined at 700 °C for 1.5 hours under nitrogen protection, washed with distilled water and anhydrous ethanol in sequence after cooling, and dried at 70 °C to obtain KOH-activated magnetic porous carbon microspheres (denoted as KOH-MPCM).

[0051] Step 5: The KOH-MPCM obtained in step 4 was mixed with selenium powder in a mass ratio of 1:0.5, and calcined at 600°C for 2 hours under nitrogen protection. After cooling, it was washed with distilled water and anhydrous ethanol in sequence, and dried at 70°C to obtain selenized magnetic porous carbon microspheres (denoted as Se-KOH-MPCM).

[0052] To compare the performance of Se-KOH-MPCM, the following materials were prepared: (1) Puffball-derived carbon microspheres: 15 g of puffball powder was calcined at 600°C for 2 h in a nitrogen atmosphere, cooled to room temperature, washed with distilled water and anhydrous ethanol, and dried at 70°C to obtain puffball-derived carbon microspheres (PCM). (2) Magnetic Fe3Se4 nanoparticles: Se-KOH-MPCM powder was calcined at 600°C for 2 h in an air atmosphere, cooled to room temperature, washed with distilled water and anhydrous ethanol, and dried at 70°C to obtain magnetic Fe3Se4 nanoparticles.

[0053] The pore structure and chemical composition of the various carbon materials prepared above were qualitatively and quantitatively analyzed using physical adsorption instrument ASAP 2020 and elemental analysis. The results are shown in Table 1.

[0054] Table 1

[0055] PCM MPCM KOH-MPCM Se-KOH-MPCM <![CDATA[Specific surface area (m 2 g -1 )]]> 35.1 67.3 619.9 266.2 <![CDATA[Total pore volume (cm 3 g -1 )]]> 0.014 0.020 0.21 0.090 Average pore size (nm) 5.1 3.8 4.98 5.4

[0056] As shown in Table 1, the specific surface area of ​​the selenized magnetic porous carbon microspheres prepared in Example 1 is 266.2 m 2 / g, total pore capacity of 0.09cm 3 / g, and an average mesopore size of 5.4nm. Although the specific surface area and total pore volume of Se-KOH-MPCM are lower than those of KOH-MPCM, the average pore size of Se-KOH-MPCM is slightly higher than that of KOH-MPCM. In particular, the specific surface area, total pore volume, and average pore volume of Se-KOH-MPCM are superior to those of MPCM and PCM. These results demonstrate that the inert atmosphere high-temperature carbonization-assisted KOH-Se dual activation technique can not only construct selenized magnetic porous carbon microspheres, but also synergistically regulate the pore structure of the carbon skeleton.

[0057] Depend on Figure 1 It can be seen that the Se-KOH-MPCM prepared in this embodiment has a microspherical structure, and its phase contains graphitized carbon, Fe3O4, Fe3Se4 and Fe3C (see Figure 2 ), the magnetic material is mainly Fe3Se4. In addition, Figure 3 It is shown that the saturation magnetization of Se-KOH-MPCM is about 15 emu g -1 , indicating that it has good magnetic separation performance.

[0058] In order to demonstrate the beneficial effects of the present invention, the Se-KOH-MPCM prepared in Example 1 was tested for its uric acid scavenging, uricase-like activity, reactive oxygen free radical scavenging, and antibacterial properties. The specific experiments are as follows:

[0059] 1. Uric acid removal performance of Se-KOH-MPCM

[0060] 20 mg of carbon material was uniformly mixed with 20 mL of a 25 mg / L uric acid aqueous solution and placed in a 37°C water bath with shaking at 120 rpm for 90 minutes. The absorbance of uric acid in the supernatant was then measured using a UV-visible spectrophotometer, and the uric acid removal capacity (mg / g) and elimination rate (%) of the carbon material were calculated according to formulas (1) and (2).

[0061]

[0062] Among them, C0 and C t is the initial concentration of uric acid and the residual concentration after clearance, V is the volume of the uric acid aqueous solution (mL), and W is the amount of carbon material used (mg).

[0063] Depend on Figure 4 It can be seen that under the same experimental conditions, the average clearance amount (16.4 mg / g) and average clearance rate (71.9%) of Se-KOH-MPCM for uric acid are better than those of MPCM (3.12 mg / g, 13.7%), KOH-MPCM (5.60 mg / g, 24.5%) and PCM (0.440 mg / g and 1.90%), indicating that the activation treatment of puffball with ammonium ferric citrate-KOH-Se can significantly improve the scavenging ability of magnetic carbon microspheres for uric acid.

[0064] 2. Factors affecting uric acid removal by Se-KOH-MPCM

[0065] (1) Effect of salt solution on scavenging performance: 20 mg of Se-KOH-MPCM was mixed with 30 mL of three different types of aqueous solutions (deionized water, PBS buffer with pH = 7.4, and 0.9 wt% NaCl aqueous solution), with the initial concentration of uric acid in each solution being 100 mg / L. The mixture was then placed in a 37°C constant temperature water bath and shaken at 120 rpm in the dark for 90 minutes. Finally, the absorbance of uric acid in the supernatant was measured, and the clearance amount and elimination rate were calculated respectively. The experimental results showed that the scavenging effect of Se-KOH-MPCM on uric acid did not change significantly in different solution systems, suggesting that the salt content in the normal physiological microenvironment of the human body hardly inhibits the scavenging effect of Se-KOH-MPCM on uric acid.

[0066] (2) Effect of solution pH on uric acid clearance performance: 20 mg of Se-KOH-MPCM was added to 30 mL of uric acid aqueous solution with different pH values ​​(5, 6, 7.4, 8, 9, 10), with the initial uric acid concentration being 100 mg / L. The solution was placed in a 37°C constant temperature water bath and shaken at 120 rpm in the dark for 90 minutes. The residual uric acid concentration in the supernatant was then measured, and the clearance amount and clearance rate were calculated. Figure 5It can be seen that when the pH of the uric acid aqueous solution is between 5.0 and 7.4, the uric acid clearance and elimination rate of Se-KOH-MPCM significantly increase with increasing pH, with the elimination rate increasing from 15.91% to 44.36%. When the pH of the uric acid aqueous solution is between 7.4 and 10, the uric acid clearance and elimination rate of Se-KOH-MPCM remain essentially constant. For practical applications, the optimal pH of the solution is 7.4.

[0067] (3) Effect of contact temperature on uric acid clearance performance: 20 mg Se-KOH-MPCM was mixed with 30 mL uric acid aqueous solution (pH = 7.4, initial uric acid concentration 100 mg / L), placed in a constant temperature water bath at different temperatures (25, 37, 40, 45 ° C), and shaken at 120 rpm in the dark for 90 min. The absorbance of uric acid in the supernatant was then measured, and the clearance amount and elimination rate were calculated respectively. The results showed that as the contact temperature of the solution increased, the clearance amount of uric acid by Se-KOH-MPCM (43.0-81.4 mg g -1 ) and clearance rate (28.9% to 55.5%) increased continuously. Considering the practical application, the optimal contact temperature was selected as 37℃.

[0068] (4) Effect of adsorbent dosage on uric acid clearance performance: Se-KOH-MPCM of different masses (15, 25, 35, 45, 55 mg) was added to 30 mL of uric acid aqueous solution (pH = 7.4, initial uric acid concentration 100 mg / L), placed in a 37°C constant temperature water bath, and shaken at 120 rpm in the dark for 90 minutes. The absorbance of uric acid in the supernatant was then measured, and the clearance amount and elimination rate were calculated. The results showed that when the adsorbent dosage increased from 0.50 mg / mL to 1.83 mg / mL, there was no significant difference in the clearance amount of uric acid by Se-KOH-MPCM (mean value was approximately 50.0 mg / g), while the elimination rate increased from 24.7% to 99.0%. Therefore, the optimal dosage of the adsorbent was 1.83 mg / mL.

[0069] (5) Effect of initial solute concentration and clearance time on uric acid clearance performance: 91.5 mg Se-KOH-MPCM was added to 50 mL of uric acid aqueous solution with different concentrations (50, 100, 200, 400, 500, 600 mg / L), placed in a 37°C constant temperature water bath and shaken at 120 rpm in the dark. Then, samples were taken at different time intervals to measure the absorbance of uric acid in the supernatant, and the clearance amount and elimination rate were calculated respectively. Figure 6Se-KOH-MPCM exhibited distinct scavenging characteristics for uric acid from aqueous solutions of varying uric acid concentrations. The removal capacity gradually increased with prolonged contact time, and higher initial concentrations required longer time to reach adsorption equilibrium. Furthermore, adsorption isotherm results demonstrated that Se-KOH-MPCM's scavenging behavior for uric acid conformed to Langmuir monolayer adsorption, with a maximum removal capacity of 239.98 mg / g.

[0070] 3. Evaluation of uricase activity

[0071] 300 μL of a 4.0 mg / mL carbon material aqueous suspension, 300 μL of a 1.0 mg / mL horseradish peroxidase aqueous solution, and 300 μL of a 3.0 mM uric acid solution (prepared in PBS buffer, pH 7.4) were sequentially added to 2.1 mL of PBS buffer, pH 7.4. The mixture was reacted in a 37°C water bath for 20 minutes. Subsequently, 2.4 mL of the supernatant was centrifuged and uniformly mixed with 300 μL of a 1 M HCl aqueous solution and 300 μL of a 60 mM 3,3'-5,5'-tetramethylbenzidine aqueous solution. The mixture was reacted at 37°C for 20 minutes, and the absorbance at 450 nm was measured. The group without carbon material was used as the blank control group.

[0072] Depend on Figure 7 It can be seen that under the same experimental conditions, compared with the blank control group, all prepared carbon materials have uricase-like activity. Among them, the average absorbance value of Se-KOH-MPCM at 450nm (A=0.1733) is much higher than that of PCM (A=0.0727), KOH-MPCM (A=0.0978), MPCM (A=0.0907), and magnetic Fe3Se4 nanoparticles (A=0.0822), indicating that Se-KOH-MPCM has the best uricase-like catalytic activity. Moreover, the uricase-like activity of Se-KOH-MPCM is much higher than that of PCM and Fe3Se4 nanoparticles, indicating that the uricase-like activity of Se-KOH-MPCM is derived from the synergistic effect of the two. Therefore, Se-KOH-MPCM is expected to achieve synergistic removal of uric acid by utilizing its multi-level pore adsorption characteristics and uricase-like function.

[0073] To further verify the cascade catalytic reaction in the process of uric acid degradation, the catalase-like enzyme of Se-KOH-MPCM can catalyze the decomposition of uric acid degradation product hydrogen peroxide to produce oxygen. The generated oxygen acts as an oxidant for the uric acid degradation reaction, further enhancing the degradation of uric acid. The experimental method is: insert the dissolved oxygen meter under the liquid surface of 30mL500mg / L uric acid aqueous solution, seal the liquid surface with liquid paraffin, and then place it on a magnetic stirrer, and adjust the speed to 350rpm / minute. Record the initial dissolved oxygen concentration, and then draw 500μL of Se-KOH-MPCM aqueous suspensions of different concentrations (Se-KOH-MPCM concentrations are 1.0mg / mL and 2.0mg / mL, respectively) and add them to the uric acid aqueous solution, and record the dissolved oxygen changes every 30 seconds. By Figure 8 Compared to the control group (500 μL of a 1.0 mg / mL Se-KOH-MPCM aqueous suspension in 30 mL of deionized water), the dissolved oxygen content of the solution gradually decreased over time after the addition of Se-KOH-MPCM to the aqueous uric acid solution. This decrease was more pronounced with increasing Se-KOH-MPCM concentration, indicating that the oxygen demand for uric acid degradation by Se-KOH-MPCM is greater than the oxygen production from its decomposition of hydrogen peroxide, ultimately manifesting as a decrease in oxygen content. These data not only demonstrate the uricase-like function of Se-KOH-MPCM but also demonstrate the existence of a cascade of catalytic reactions during further uric acid degradation.

[0074] 4. Performance of scavenging active nitrogen / oxygen free radicals

[0075] Patients with acute gout, renal failure, or organ failure often experience an imbalance in oxidative stress levels. This physiological phenomenon is most directly manifested by abnormally elevated levels of reactive oxygen and nitrogen species (ROS / RNOs). This symptom is exacerbated during hemoperfusion. Therefore, hemoperfusion adsorbents that effectively regulate oxidative stress while removing toxins from the blood are crucial for clinical applications. To address this, the scavenging properties of Se-KOH-MPCM and control materials for RNO / RNO free radicals were investigated.

[0076] DPPH scavenging experiment: 100 μL of 3.0 mg / mL carbon material aqueous suspension was thoroughly mixed with 900 μL of 0.1 mM DPPH aqueous solution, and the mixture was allowed to react at 37°C for 10 minutes. The mixture was centrifuged at 10,000 rpm for 2 minutes, and the absorbance of the supernatant at 520 nm was measured. The sample without carbon material was used as the control group. Figure 9 As shown in the results, PCM had almost no scavenging effect on DPPH, while KOH-MPCM and Se-KOH-MPCM had similar scavenging abilities on DPPH, with a scavenging rate of up to 31.6%, indicating that Se-KOH-MPCM is expected to exhibit good antioxidant efficacy during blood perfusion.

[0077] ABTS free radical scavenging experiment: 100 μL of 3.0 mg / mL carbon material aqueous suspension was mixed with 900 μL of ABTS free radical (the absorbance of the ABTS free radical dilution was about 0.8) aqueous solution, placed in a 37°C water bath for 10 minutes, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was taken to measure the absorbance value at 734 nm. Figure 10 As shown in the results, the scavenging ability of KOH-MPCM (73.7%) and Se-KOH-MPCM (64.4%) on ABTS free radicals was significantly higher than that of MPCM (20.2%) and PCM (4.9%).

[0078] Superoxide radicals (O2 - ) Clearance experiment: 150 μL of a 6.0 mg / mL carbon material aqueous suspension, 150 μL of a 0.4 mM riboflavin aqueous solution, 150 μL of a 250 mM L-methionine aqueous solution, and 150 μL of a 1.5 mM nitro blue tetrazolium aqueous solution were sequentially added to 2.4 mL of a pH 7.4 PBS buffer solution. The mixture was irradiated with a xenon lamp at a constant temperature of 37°C for 10 minutes, and the absorbance of the supernatant was measured at 560 nm. Figure 11 As shown in the results, the scavenging performance of Se-KOH-MPCM (37.8%) on superoxide radicals was significantly better than that of KOH-MPCM (29.1%), MPCM (18.6%) and PCM (10.9%).

[0079] H2O2 scavenging experiment: Catalase (CAT) can catalyze H2O2 to generate O2, and the oxygen content produced by the decomposition of H2O2 by CAT enzyme can be monitored by a dissolved oxygen meter. If Se-KOH-MPCM has intrinsic hydrogen peroxide-mimicking enzyme catalytic activity, it can achieve the removal of endogenous highly expressed H2O2 and H2O2, the product of uric acid decomposition by uricase-like enzymes, during the blood perfusion process, thereby alleviating the level of oxidative stress damage. The specific experimental method is: 500μL of 4.0mg / L carbon material water suspension and 0.5mL30wt% H2O2 aqueous solution are successively added to 49mL of PBS buffer solution with pH=7.4. After the paraffin is used to seal the liquid surface layer, the dissolved oxygen content is measured every 30 seconds using a dissolved oxygen meter, and the ΔOD value is calculated. Figure 12 As shown, the CAT activities of Se-KOH-MPCM (3.88 mg / L) and KOH-MPCM (3.70 mg / L) were higher than those of PCM (1.14 mg / L) and MPCM (2.04 mg / L).

[0080] 5. Antibacterial properties

[0081] The antibacterial test was carried out using the plate counting method. The specific method was as follows: 0.1 mL 10 4 CFU mL -1Escherichia coli or Staphylococcus aureus solutions were mixed with 0.9 mL of Se-KOH-MPCM aqueous suspensions of varying concentrations and incubated at 37°C for 4 hours. A 50 μL aliquot of the mixed bacterial solution was inoculated onto solid culture medium and incubated in a 37°C incubator for 24 hours. The colonies were counted and the inhibition rate was calculated. Figure 13 and Figure 14 The results showed that Se-KOH-MPCM had an antibacterial effect on both Escherichia coli and Staphylococcus aureus. As the concentration of Se-KOH-MPCM increased, the average colony counts of Escherichia coli and Staphylococcus aureus decreased significantly, and the antibacterial effect gradually increased. When the concentration of Se-KOH-MPCM was 2 mg mL -1 When the concentration of Se-KOH-MPCM was 50.004mol / L, the inhibition rates against Escherichia coli and Staphylococcus aureus were 80.95±2.04% and 55.73±2.09%, respectively, indicating that Se-KOH-MPCM has a good inhibitory effect on both Gram-negative and Gram-positive bacteria.

[0082] 6. Selectivity of Se-KOH-MPCM in removing uric acid

[0083] During the blood perfusion process, if the adsorbent adsorbs plasma proteins, it will lead to thrombocytopenia and easily cause coagulation complications. Therefore, it is extremely important for the adsorbent to have good anti-protein adhesion properties during the blood perfusion process. In this experiment, bovine serum albumin (BSA) was used as the research model. 10mg Se-KOH-MPCM was mixed with 2mL 2.0mg mL -1 BSA was mixed with PBS buffer (pH 7.4) and incubated in a 37°C water bath for 1 hour. To quantitatively evaluate the protein adhesion capacity of Se-KOH-MPCM, the BSA-adsorbed carbon material was first washed with PBS buffer and deionized water, then shaken for 2 hours in a washing solution (50 mM NaOH aqueous solution containing 2.0 wt% sodium dodecyl sulfate) at 37°C to desorb BSA. The protein concentration in the washing solution was determined using a BCA kit, and the amount of protein adsorbed was calculated. The results showed that under the same experimental conditions, the average BSA adsorption capacity of medical activated carbon (54 μg / g) was significantly higher than that of Se-KOH-MPCM (5.8 μg / g) and MPCM (15 μg / g), indicating that Se-KOH-MPCM has good anti-protein adsorption capacity and performs better than clinical medical activated carbon.

[0084] 7. Renewability and desorption of uric acid by Se-KOH-MPCM

[0085] Reusability experiment: Weigh 45 mg of Se-KOH-MPCM in a conical flask and add 30 mL of 100 mg L -1The collected Se-KOH-MPCM was washed with 20 mL of 1 mg mL uric acid solution (PBS buffer solution with pH = 7.4 as solvent) and shaken in a 37°C water bath for 1.5 hours. The absorbance of the supernatant was measured. -1 The NaOH aqueous solution was shaken for 30 minutes, then washed with deionized water until neutral and dried for later use. The washed Se-KOH-MPCM was weighed and the above process repeated four times to calculate the adsorption capacity. The results showed that after five cycles, the Se-KOH-MPCM still maintained a high uric acid clearance rate of 74.57%, demonstrating the excellent reproducibility of the Se-KOH-MPCM. Notably, the excellent reusability of the Se-KOH-MPCM could effectively reduce the cost of hemoperfusion therapy for acute gouty kidneys.

[0086] Desorption experiment: Weigh 20 mg Se-KOH-MPCM and add 20 mL 100 mg L -1 A uric acid solution (PBS buffer, pH 7.4) was shaken at 37°C for 4 hours. The precipitate was collected by magnetic separation and dried. It was then added to 10 mL of deionized water and shaken at 37°C. The absorbance of the solution was measured at 15, 30, 60, 90, 150, and 270 minutes. The calculation formula is as follows:

[0087]

[0088] η des is the desorption rate, C0 is the concentration before adsorption (mg / L), C ads is the concentration after adsorption (mg / L), C des is the concentration of the solution after desorption (mg / L).

[0089] The results showed that Se-KOH-MPCM released almost no uric acid (η des =0.33%), indicating that Se-KOH-MPCM has a strong binding force with uric acid molecules and will not cause the secondary release of uric acid during blood perfusion.

[0090] 8. The scavenging performance of Se-KOH-MPCM on other toxins

[0091] Acute or refractory gouty kidney is often accompanied by problems such as decreased renal function. Furthermore, patients with gouty kidney often suffer from urinary tract infections and may take antibiotics, but antibiotics are primarily metabolized by the kidneys, which can increase the burden on the kidneys. Therefore, if a patient with gouty kidney experiences worsening symptoms after taking antibiotics, prompt removal of antibiotics from the blood is essential. Therefore, in addition to their efficient clearance of uric acid, hemoperfusion adsorbents are also particularly important for the removal of common antibiotics and hepatotoxicants. To investigate the clearance properties of Se-KOH-MPCM for common toxins and antibiotics associated with gouty kidney failure, clearance experiments were conducted using common antibiotics (such as norfloxacin, doxycycline, chlortetracycline, and oxytetracycline) and hepatotoxicants (indole, bilirubin, creatinine, and urea) as models. The test method is as follows: 30mg Se-KOH-MPCM was mixed with 30mL 100mg / L different toxin solutions, placed in a 37℃ constant temperature water bath and shaken at 120rpm for 1.5 hours, and the absorbance of the corresponding toxin before and after adsorption was measured using a UV-visible spectrophotometer. The adsorption amount of the corresponding toxin molecule was calculated using the standard curve of each toxin molecule. Figure 15 ) and the results of liver and kidney toxin clearance ( Figure 16 ) showed that Se-KOH-MPCM can adsorb a variety of different types of toxin molecules, showing universal applicability. It was further demonstrated that Se-KOH-MPCM not only effectively removes excess uric acid in patients with acute gouty kidney, but also has a strong clearance effect on common antibiotics and hepatotoxins, making it a promising adjunct treatment for acute gouty kidney.

[0092] 9. Blood compatibility test

[0093] Hemocompatibility is a key indicator of hemoperfusion adsorbents. Rupture of red blood cells releases hemoglobin, causing anemia and even organ damage, making hemolysis rate determination extremely important. The test method is as follows: 2.0 mL of anticoagulated rat whole blood is centrifuged at 3000 rpm for 5 minutes, leaving approximately 1.0 mL of red blood cells. 3 mL of PBS buffer (pH 7.4) is added, and the mixture is centrifuged at the same speed for 5 minutes. The supernatant is aspirated, leaving the red blood cell pellet, and the washing is repeated three times. Approximately 1.0 mL of red blood cells is mixed with 50.0 mL of PBS buffer to prepare a diluted red blood cell suspension (2% v / v). To 1.5 mL of the red blood cell suspension is added 0.5 mL of an aqueous suspension of Se-KOH-MPCM at various concentrations (1, 2, 4, and 5 mg / mL). PBS buffer is used as a negative control, and deionized water is used as a positive control. The mixture is incubated at 37°C for 1 hour. After incubation, the mixture is centrifuged, and the absorbance of hemoglobin in the supernatant is measured at 540 nm to calculate the hemolysis rate. The results showed that the final concentration of Se-KOH-MPCM was 0.25 mg mL -1 Increase to 1.25 mg mL -1Still no hemolysis occurs, and the hemolysis rate is less than 5%, indicating that Se-KOH-MPCM has good blood compatibility.

[0094] 10. Evaluation of Clearance Performance of Simulated Hemoperfusion

[0095] To simulate the hemoperfusion process used in clinical treatment of acute gouty kidney, a dynamic clearance experiment was conducted using Se-KOH-MPCM as an adsorbent. The perfusion apparatus consisted of a simulated solution of pig blood containing toxins (uric acid, creatinine, and urea), a chromatography column loaded with Se-KOH-MPCM, a peristaltic pump, and a water bath. 150 mg of Se-KOH-MPCM was added to the chromatography column, and 48.0 mL of a toxin-simulating solution (final concentrations of 70 mg / L uric acid, 100 mg / L creatinine, and 300 mg / L urea) was added to an Erlenmeyer flask. The Erlenmeyer flask containing the toxin-simulating solution was connected to the peristaltic pump and chromatography column to form a closed loop. The peristaltic pump operated at a flow rate of 2 mL / min to conduct toxin clearance experiments. The mixed solution in the Erlenmeyer flask was sampled at regular intervals for uric acid, creatinine, and urea. The absorbance of the remaining toxins in the solution was measured, and the clearance rate was calculated. Figure 18 and Figure 19 The results showed that after 3 hours of simulated perfusion in the pig blood system, the clearance rate of uric acid by Se-KOH-MCPM was close to 100%, indicating that Se-KOH-MPCM has a good clearance ability for uric acid in the pig blood system; at the same time, its clearance rate for creatinine was close to 30%; on the contrary, Se-KOH-MPCM had almost no clearance effect on urea.

[0096] In summary, the Se-KOH-MPCM prepared by this invention not only exhibits uricase- and peroxidase-like activities but also possesses toxin scavenging and oxidative stress alleviation functions. Furthermore, studies have confirmed that Se-KOH-MPCM possesses both good antibacterial properties and excellent blood compatibility, providing a theoretical basis for in vivo hemoperfusion therapy for acute gouty kidney and, in particular, a new option for the clinical application of low-cost, multifunctional hemoperfusion adsorbents.

Claims

1. A method for preparing selenized magnetic porous carbon microspheres for blood perfusion, characterized in that The method consists of the following steps: Step 1: Peel the puffball, take the inner fluff, wash and dry it to obtain puffball powder; Step 2: Dissolve the iron salt in distilled water, add the puffball powder from step 1, stir at room temperature for 2 to 4 hours, and then heat-induce the assembly reaction at 60 to 90°C for 12 to 48 hours; Step 3: calcining the product obtained in step 2 at 550-700° C. for 1-3 hours under an inert gas atmosphere, washing the product with distilled water and anhydrous ethanol, and drying the product to obtain puffball-derived magnetic carbon microspheres; Step 4: mixing the puffball-derived magnetic carbon microspheres in step 3 with KOH in a mass ratio of 1:0.1 to 1:3, calcining at 500 to 800° C. for 1 to 3 hours under inert gas protection, washing with distilled water and anhydrous ethanol in sequence, and drying to obtain KOH-activated magnetic porous carbon microspheres; Step 5: The KOH-activated magnetic porous carbon microspheres in step 4 are mixed with selenium powder in a mass ratio of 1:0.2 to 1:2, calcined at 550 to 700° C. for 1 to 3 hours under inert gas protection, washed with distilled water and anhydrous ethanol in turn, and dried to obtain selenized magnetic porous carbon microspheres.

2. The method for preparing selenized magnetic porous carbon microspheres for blood perfusion according to claim 1, characterized in that: In step 2, the mass ratio of iron (III) in the iron salt to puffball powder is 1:3 to 1:9; the concentration of the iron salt added to the distilled water is 0.6 to 1.8 mol / L.

3. The method for preparing selenized magnetic porous carbon microspheres for blood perfusion according to claim 1, characterized in that: In step 2, the iron salt is any one of ammonium ferric citrate, anhydrous ferric chloride, ferric ammonium oxalate, and ferric nitrate nonahydrate.

4. The method for preparing selenized magnetic porous carbon microspheres for blood perfusion according to claim 1, characterized in that: In step 2, the assembly reaction was heat-induced at 70°C for 24 hours.

5. The method for preparing selenized magnetic porous carbon microspheres for blood perfusion according to claim 1, characterized in that: In step 3, the product obtained after the reaction in step 2 is calcined at 600° C. for 2 hours under the protection of an inert gas.

6. The method for preparing selenized magnetic porous carbon microspheres for blood perfusion according to claim 1, characterized in that: In step 4, the puffball-derived magnetic carbon microspheres prepared in step 3 were mixed with KOH in a mass ratio of 1:2 and calcined at 700° C. for 1.5 hours under inert gas protection.

7. The method for preparing selenized magnetic porous carbon microspheres for blood perfusion according to claim 1, characterized in that: In step 5, the KOH-activated magnetic porous carbon microspheres in step 4 were mixed with selenium powder in a mass ratio of 1:0.5, and calcined at 600° C. for 2 hours under inert gas protection.

8. The method for preparing selenized magnetic porous carbon microspheres for blood perfusion according to claim 1, characterized in that: In steps 3, 4 and 5, the inert gas is nitrogen or argon.

9. Selenized magnetic porous carbon microspheres for blood perfusion obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the selenized magnetic porous carbon microspheres according to claim 9 as an adsorbent in blood perfusion.

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